PCR Troubleshooting and Optimization

PCR Troubleshooting
and Optimization
The Essential Guide
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Caister Academic Press
Copyright 201 1
Caister Academic Press Norfolk, UK
wwwcai stercom
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Smli *OU7P2*
Magic in Solution: an Introduction and Briet
History of PCR
Carl T. ittwer and !ared s "arrar
A#stract
2he polymerase chai% reactio% 6PC97 has #ecome a 1$%dame%tal tool i% molec$lar research a%d cli%ical testi%g 2he
origi%s of PC9 a%d its early e)ol$tio% are descri#edC i%cl$di%g adaptatio% to 9NAC thermosta#le polymerases,
a$tomatio%, impro)eme%ts i% speci1kity a%d rapid temperat$re cycli%g Perhaps the most sig%ihca%t ad)a%ce is real&time
PC9, com#i%i%g #oth amplihcatio% a%d detectio% i%to o%e i%str$me%t as a s$perior sol$& tio% for %$cleic acid
3$a%tihcatio% 9eal&time PC9 is e%a#led #y mo%itori%g the reactio% with do$#le stra%ded 'NA dyes or speci1(c pro#es,
i%cl$di%g hyQrolysis, hy#ridiDatio% a%d co%formatio%&se%siti)e pro#es Farly real&time *%str$me%ts are compared PC9
prod$ct a%d pro#e melti%g a%alysis co%ti%$es to impro)e i% resol$tio%, allowi%g greater se3$e%ce detai4 for ge%otypi%g
a%d )aria%t sca%%i%g <icro1l$idic plat1Mrms a%d digital PC9 are desti%ed to fi%d more applicatio%s i% the 1$t$re
Origins of PCR
Iith R #illio% #ases i% the h$ma% ge%ome, it is %ot easy to fi%d a%d a%alyse the small se3$e%ce regio%s that co%hrm a
ge%etic disorder, ide%tify a% o%coge%ic cha%ge or detect micro#ial i%fectio% 2he polymerase chai% reactio% 6PC97
pro)ides this foc$s Si%ce its de)elopme%t 20 years ago, it has #ecome the most importa%t tool for ))orki%g with %$cleic
acids i% molec$lar #iology a%d cli%ical diag%ostics *t deser)es s$ch Ce%tral recog%itio% #eca$se ofits simplicity
!efore PC9, molec$lar methods were m$ltistepped, la#orio$s a%d time&co%s$mi%g 2o amplify 'NA, it had to #e
clo%ed i%to plasmids, the plasmids i%serted i%to #acteria, the #acteria grow% i% c$lt$re, the #acteria har)esteQ, the
plasmids isolated from the #ac& teria, a%d the 'NA i%serts separated from the plasmid 'NA So$ther% #lotti%g re3$ired
m$ltiple steps of restrictio% e%Dyme digestio%, electrophoresis, #lotti%g o%to mem#ra%es, hy#ridiDatio% with
radioacti)ely la#elled oligo%$cleotide pro#es a%d de)elopme%t o% L&ray film 2hese early tech%i3$es re3$ired large
amo$%ts of 'NA a%d stro%g tech%ical eNpertise for co%siste%t res$lts
<a%y of the compo%e%ts %ecessary for PC9 were a)aila#le #e1ore its i%)e%tio% i% the 1,.0s #y Kary <$llis a%d
colleag$es at the Cet$s Corporatio% 6<$llis et al, 1,.SC Saiki et al, 1,.07 2he sy%thesis of 'NA oligo%$cleotides had
#ee% per1ected a%d strides towards its a$tomatio% were $%der way 6Agarwal et al, 1,-07 2he Sa%ger method for
se3$e%ci%g,
Accumulating
Products
))hich wo% the No#el PriDe i% 1,.0C demo%strated 'NA polymerase eNte%sio% of si%gle
oligo%$cleotide primers a%%ealed to their compleme%tary stra%ds 6Sa%ger et al, 1,--7 PC9
com#i%ed aspects of these de)elopme%ts i%to a two primer mediated amplihcatio% of a specific
'NA se3$e%ce
PC9 re3$ires template 'NAC a 'NA polymeraseC two se3$e%ce speci1(c primers a%d
deoNyri#o%$cleoside triphosphates 6dN2Ps7 2hese compo%e%ts are cycled i% sol$tio% thro$gh
three temperat$res correspo%di%g to de%at$ratio%C a%%eali%g a%d eNte%sio% 6=ig 117
'e%at$ratio% separates the do$#le stra%ded temp4ate 'NA i%to si%gle stra%ds which the%
hy#ridiDe to specihc primers d$ri%g the a%%eali%g step FNte%sio% allows the polymer& ase to
i%corporate dN2Ps i%to the eNte%di%g RT e%d of the primer compleme%tary to the template
'NA A1ter R0 temperat$re cycleSC the 'NA template #o$%ded #y the primers is amplihed a
millio%& to a #illio%&folQ *%stead of relyi%g o% a compleN in vivo process s$ch as clo%i%gC the
replicatio% of 'NA was per1ormed in vitro. PC9 greatly red$ced the %$m#er of steps re3$ired to
ge%erate apprecia#le 3$a%tities of 'NA %ecessary for ma%y dow%stream applicatio%s 2he
accepta%ce of PC9 i% the scie%ti1k comm$%ity was relati)ely swiftC with a% i%depe%de%t
research gro$p $si%g the tech%i3$e withi% a year 68erlaa%&de 8ries et al, 1,.S7 PC9 has si%ce
re)ol$tio%iDed molec$lar #iology a%d cli%ical diag%osticSC ear%i%g Kary <$llis the No#el
PriDe i% 1,,R
Early PCR eolution
Initial Template !enaturation
"igure #$# T$e PCR cycle. Initial tem%late &'A is denatured #y $eat.
T$e reaction is t$en cooled to anneal two oligonucleotide %rimers(
eac$ annealing to o%%osite strands wit$ t$eir )*+ends %ointed
in,,ard to,,ard eac$ ot$er. A %olymerase t$en e-tends eac$
%rimed tem%late to dou#le t$e amount of targeted &'A. T$e cycle
is re%eated ./+0/ times t$roug$ successi,e ste%s of denaturation(
annealing and e-tension. E,entually if reagents are not limiting(
t$e concentration of accumulating Products #ecomes $ig$ enoug$
t$at dou#le strand %roduct tormation is ta,oured o,er %rimer
annealing( and t$e reaction %lateaus.
Magic in Solution: an Introduction and Briet History of PCR I
PC9 is simple a%d elega%t Ut is remarka#ly ro#$st a%d tolerates the additio% of ma%y di& )erse
reage%ts s$ch as electrophoresis dyes 6Iittwer a%d 5arli%gC 1,,17 PC9 has spaw%ed
i%%$mera#le modihcatio%s a%d ma%y related tech%i3$es ?owe)er
C
if it ))ere %ot for a seriesof
e%ha%ceme%ts o)er the years, it might ha)e remai%ed a la#oratory c$riosity 9e)erse
tra%scriptase PC9, thermosta#le polymerases, a$tomatio%, impro)ed specificity, a%d rapid
temperat$re cycli%g were early de)elopme%ts i% the e)ol$tio% of PC9 'issemi%atio% of PC9
was hi%dered #y restricti)e pate%t policies, partic$larly lice%ce fees for cli%ical la#ora& tories
that eNtracted 12&20V of cli%ical testi%g re)e%$es ?o))e)er, e)e% $%derthis lia#ility, PC9
ofte% pro)ed to #e the #est sol$tio% as a 1leNi#le a%d ro#$st method for a )ariety of
applicatio%s, res$lti%g i% ma%y la#oratory&de)eloped tests
Re,erse transcri%tase PCR
2he disco)ery of a% W9NA&Qepe%Qe%t 'NA polymeraseX, or re)erse tra%scriptase, was i%itially
reported #y two i%depe%de%t gro$ps 62emi% a%d <iD$ta%i, 1,-07 2he s$gges& tio% of s$ch a%
e%Dyme was %ew a%d we%t agai%st the widely accepted WCe%tral 'ogmaX propo$%ded #y
Iatso% a%d Crick of 'NA #ei%g tra%scri#ed i%to 9NA a%d the% tra%slated i%to protei% *t was
there1ore met with miNed feeli%gs i% the scie%ti1(c comm$%ity ?owe)er, the co%cept gai%ed
wider accepta%ce after its i%depe%de%t co%1:matio% 65ree% et al, 1,-0C Spiegelma% et al, 1,-0a&
c7, a%d i% 1,-0 2emi% a%d !altimore were @oi%tly awarded the No#el PriDe for their disco)ery
*t did %ot take lo%g a1ter the disco)ery of PC9 for its power to #e $sed to st$dy tra%scrip&
tio% le)els i% )ario$s tiss$es 6!ecker&A%dre a%d ?ahl#rock, 1,.,C Ia%g et al, 1,.,7 2he
amo$%t of 9NA re3$ired for PC9 was m$ch less tha% that re3$ired for other assays s$ch as
%orther% #lotti%g, %$clease protectio% assays or in situ hy#riQiDatio% PC9s a#ility to amplify
low copy %$m#er tra%scripts made it immediately applica#le for detectio% of low copy m9NAs
a%d m9NA hom a small %$m#er of cells or a small amo$%t of tiss$e sample
2raditio%al re)erse tra%scriptase PC9 is a $sef$l tech%i3$e for 3$a%tihcatio% of m9NA
tra%scriptio% ?owe)er, i% terms of relati)e or a#sol$te 3$a%tihcatio%, traditio%al re)erse
tra%scriptase PC9 6as opposed to real&time 92&PC97 falls short #eca$se of the e3$aliDi%g e1Kect
of the PC9 platea$ Small di1Kere%ces i% the i%itial co%ce%tratio% of templates are %ot easily
discer%ed #y simple e%d&poi%t stai%i%g PC9 ))ith a competiti)e template is o%e sol$tio%, with
the relati)e amo$%ts of target a%d competitor o1te% disti%g$ished #y siDe, #$t the method
re3$ires either prior k%owledge of the approNimate template co%ce%tratio%, or m$ltiple
reactio%s at differe%t co%ce%tratio%s 2oday, real&time, re)erse tra%scriptase, 3$a%titati)e PC9
63PC97 with SY!9 5ree% * is the most commo% method for tra%script 3$a%tihcatio% #eca$se
ofsimplicity a%d cost 6<orriso% et al, 1,,.7
T$ermosta#le %olymerases
2he earliest reports of PC9 $sed a modihed )ersio% of 'NA polymerase 1 hom E. coli k%ow% as
the Kle%o)) hagme%t 6Kle%ow a%d ?e%%i%gse%, 1,-0C <$llis a%d =aloo%a, 1,.-C Saiki et al,
1,.07 2ho$gh this e%Dyme prod$ces do$#le stra%ded 'NA hom primed si%gle stra%ded
template at R-Zc, it is i%acti)ated at the high temperat$res %ecessary for the de%at$ratio% of
ge%omic 'NA 2he 1(rst PC9s were per1ormed i% three water #aths set to three temperat$res
for de%at$ratio%, a%%eali%g a%d eNte%sio% 9eactio%s were ma%$ally tra%s1erred hom o%e water
#ath to the %eNt i% the cyclic ma%%er esse%tial for PC9 Hwi%g to the thermola#ile %at$re of the
Kle%ow hagme%t, hesh e%Dyme had to #e added to the reactio% miNt$re after each de%at$ratio%
step
A pote%tial sol$tio% to the pro#lem of heat&i%d$ced e%Dyme de%at$ratio% was disco)& ered
1/ I ittwer and
lo%g #e1ore the creatio% of PC9 2homas ' !rock a%d ?$dso% =reeDe, micro#iologists
worki%g at the hot spri%gs of Yellowsto%e Natio%al Park, disco)ered a hyperthermophilic
#acteri$m, Thermus aquaticus, which thri)ed at high temperat$res 6!rock a%d =reeDe, 1,S,7 *%
1,-S, Chie% a%d colleag$es, at the U%i)ersity ofCi%ci%%ati, reported o% the isola& tio% a%d
p$rihcatio% of a 'NA polymerase from Thermus aquaticus, %oti%g its optim$m e%Dymatic acti)ity
of .0Zc
*t was %ot $%til 1,..,12 years a1ter its isolatio%, that the 1(rst report of PC9 $si%g hermus
aquaticus (Taq7 polymerase was p$#lished 6Saiki et al, 1,..7 !efore Taq polymerase, PC9 was
time&co%s$mi%g a%d i%efficie%t #eca$se the e%Dyme had to #e reple%ished after each
de%at$ratio%, re3$iri%g i% aggregate a large amo$%t of e%Dyme Iith a thermosta#le poly&
merase, %o additio% steps were %ecessary #eca$se the e%Dyme retai%ed acti)ity each cycle
=$rthermore, higher a%%eali%g a%d eNte%sio% temperat$res co$ld #e $sed, i%creasi%g the
e1hcie%cy, se%siti)ity a%d speed of the reactio% F1hcie%cy was i%creased #eca$se a%%eali%g a%d
eNte%sio% were less a1Kected #y seco%dary str$ct$res at higher temperat$res Se%siti)ity was
i%creased #eca$se higher primer a%%eali%g temperat$res res$lted i% more stri%ge%t
hy#ridiDatio% =i%ally, the smaller spread #etwee% a%%eali%g a%d de%at$ratio% tempera& t$res,
a%d the high eNte%sio% rate of Taq polymerase #et))ee% -0Zc a%d .0Zc pro)ided the pote%tial
for rapid cycli%g
Automation
!e1ore thermosta#le polymerases were $sed i% PC9, thermal cyclers were $%wieldy
*%str$me%ts with i%tegrated 1l$idics to add fresh e%Dyme after each de%at$ratio% Taq
polymerase greatly red$ced the e%gi%eeri%g compleNity of thermal cyclers, re3$iri%g o%ly
temperat$re cycli%g #$t %ot li3$id ha%dli%g *t did %ot take lo%g #e1ore a )ariety of thermal
cycli%g Sol$tio%s appeared *%str$me%ts progressed rapidly from la#oratory oddities to
mai%stream commodities Some early homemade eNamples cha%ged the temperat$re of
statio%ary reac& tio%s with flowi%g water or ro#otically tra%s1erred samples #et))ee% co%sta%t
temperat$re water #aths 6=o$lkes et al, 1,..C Ieier a%d 5ray, 1,..7 ?owe)er, water has some
draw& #acks Hwi%g to its large thermal mass a great amo$%t of e%ergy a%d time is re3$ired to
heat or cool water to a speci1ic temperat$re *% co%trast, air has a )ery low thermal mass a%d
was $sed i% some Systems 6Iittwer et a. 1,.,7 <a%y thermal cyclers %o))$se Peltier
eleme%ts a%d metal #locks for heati%g a%d cooli%g 6Collasi$s et al, 1,.,C Iitt#rodt a%d Frhardt,
1,.,7
2oday, PC9hardware a%d reage%ts are commo%place i% research a%d diag%ostic la#ora&
tories 2he *%str$me%ts ha)e e)ol)ed to fill a )ariety of #atch siDe a%d time&to&res$lt %eeds
2hermal cycli%g co%cer%s %ow foc$s o% iss$es of speed, temperat$re $%iformity, sample
)ol$me a%d i%creased thro$ghp$t <a%y thermal cycli%g Sol$tio%s, heat&sta#le polymer& ases,
a%d commercial PC9 mastermiNes that i%cl$de all compo%e%ts eNcept primers a%d template
'NA are a)aila#le commercially
2he %eNt #ig step i% PC9 a$tomatio% was co%%ecti%g the amplihcatio% a%d detectio% stages
to co%trol PC9 prod$ct co%tami%atio% "a#oratories ca% #e plag$ed #y false&positi)e res$lts if
prod$cts from a prior reactio% fi%d their way i%to a 1$t$re reactio% with the same primers 2his
co%tami%atio% is $s$ally co%trolleQ #y separati%g pre& a%d post&amplificatio% processes a%d
care1$l atte%tio% to reactio% preparatio% 6Kwok a%d ?ig$chi, 1,.,7 A%other sol$tio% is to
a$tomate #oth ampli14catio% a%d detectio% i% a closed&)essel System, elimi%at& i%g PC9prod$ct
eNpos$re to the e%)iro%me%t 6=i%dlay et al, 1,,R7 2he #est sol$tio% is to amplify a%d a%alyse at
the same time #y real&time PC9 a%d[or melti%g a%alysis
S%eci2icity
PC9 speci14city $%der ideal co%ditio%s is eNtraordi%ary ?owe)er, the h$ma% ge%ome is )ery
large a%d primers may #i%d %ot o%ly to their i%te%ded target #$t also to other areas of the
ge%ome =$rthermore, the primers i% PC9 are at high co%ce%tratio%s, so e)e% mi%or self or
cross compleme%tatio% may i%itiate primer dimers 2hese side reactio%s ca% create so&called
%o%&specificX prod$cts other tha% the desired prod$ct A %$m#er of methods to pre)e%t what
Kary <$llis termed Wcold oligodeoNyri#o%$clear f$sio%X 6<$llis, 1,,17 ha)e #ee% de)eloped to
pre)e%t primer eNte%sio% at low 6room7 temperat$res where polymerase acti)ity, altho$gh
greatly red$ced, is still capa#le of eNte%di%g primers
2he first Whot&startX tech%i3$es relied o% addi%g a% esse%tial reactio% compo%e%t 6s$ch as
the polymerase7 o%ly a1ter the reactio% had reached high temperat$res to 1a)o$r specific primer
a%%eali%g 6'\3$ila et al, 1,,17 2his re3$ired ope%i%g the reactio% Co%tai%er a%d i%creased the
possi#ility of PC9 co%tami%atio% 2o circ$m)e%t this pro#lem, waNes a%d greases were $sed to
physically partitio% reage%ts ))ith a #arrier that ))o$ld melt at high temperat$re, miNi%g the
esse%tial reage%t6s7 with the other reactio% compo%e%ts 6Cho$ et al, 1,,27
*%stead ofphysical separatio%, polymerase acti)ity ca% #e i%hi#ited at room temperat$re
=or eNample, mo%oclo%al a%ti#odies agai%st the acti)e site of the po4ymerase ca% i%hi#it the
e%Dyme $%til they de%at$re at high temperat$re 6Kellogg et al, 1,,/7 Alter%ati)ely, the
polymerase acti)e site ca% #e chemically modihed #y heat&la#ile co)ale%t modi1(catio%s that
#reak dow% a%d acti)ate the e%Dyme at high temperat$re 6!irch, 1,,S7 *%stead of i%acti)ati%g
the polymerase, oligo%$cleotide primers or dN2Ps ca% #e modihed at their RT&e%d with similar
heat&la#ile li%kages 6Ko$khare)a et al, 200.C "e#ede) et al, 200.7
<a%y diAere%t reage%ts are %ow a)aila#le to a$gme%t PC9 specihcity, #$t they are $s$ally
o%ly %ecessary whe% the template copy %$m#er is low Ne)ertheless, s$ch reage%ts are a% easy
way to i%crease the ro#$st%ess of PC9, sometimes maki%g optimiDatio% $%& %ecessary *f a hot
start method is re3$ired, the #est method depe%ds o% the circ$msta%ces =or eNample, a%
a%ti#ody&mediated hot&start is more $se1$l i% rapid PC9 #eca$se chemi& cally modihed
polymerases typically re3$ire 10&R0 mi% for acti)atio%, lo%ger tha% a% e%tire rapid&cycle
PC9protocol
Ra%id tem%erature cycling
Ihe% PC9 1(rst #ecame pop$lar i% the late 1,.0s the process was slow A typical a$tomated
protocol was 1 mi% for de%at$ratio% at ,/Zc, 2 mi% for a%%eali%g at 334c( a%d R mi% for eN&
te%sio% at -2Zc Iith temperat$re tra%sitio%s i%cl$ded, . mi% cycles ))ere typical re3$iri%g / l(
for R0 cycles No o%e k%ew how r%$ch time was %ecessary for each stage Cycle times were
slow #eca$se of the large )ol$mes 6100 ), co%tai%ers 6micro1$ge t$#es7, a%d heaters 6metal
#locks7 commo% at the time Usi%g samples i% capillary t$#es a%d temperat$re co%& trol with
circ$lati%g air, 10 mi% PC9 6R0 cycles of 20 s eacl(7 soo% demo%strated that these physical
limitatio%s ))ere %ot #iochemical 6Iittwer et al, 1,,07 9apid&cycle protocols $se mome%tary
or W0T s holds at the de%at$ratio% a%d a%%eali%g temperat$res, showi%g that de%at$ratio% a%d
a%%eali%g occ$r )ery 3$ickly if the temperat$re is acc$rately co%trolled 2he time a%d
temperat$re re3$ireme%ts for each PC9 stage were the% esta#lished 6Iittwer a%d5arli%g,
1,,17 $si%g rapid&cycle i%str$me%tatio% 6=ig 127
2erms s$ch as WrapidX or WfastX are relati)e a%d )ag$e A 1 h PC9 is fast compared to /
ho$rs, #$t slow compared to 10 mi% =$rthermore, 1aster PC9 is possi#le if yo$ start with a
1. I ittwer and
higher template co%ce%tratio% or $se fewer cycles *t is #etter to defi%e the time re3$ired for
each cycle a%d rapid&cycle PC9 has #ee% deh%ed as R0 cycles i% 10&R0 mi% 6Iittwer et al,
1,,/7 so that each cycle is 20&S0 s each 2he act$al time of each cycle is lo%ger tha% the s$m
of the times programmed for de%at$ratio%, a%%eali%g a%d eNte%sio% *%deed, d$ri%g rapid PC9
the temperat$re is $s$ally cha%gi%g 2his challe%ges the e3$ili#ri$m paradigmX of PC9, where
three reactio%s 6de%at$ratio%, a%%eali%g a%d eNte%sio%7 occ$r at three temperat$res o)er three
time periods each cycle 6=ig 1R, le1t7 Altho$gh i%t$iti)e, the e3$ili#ri$m paradigm does %ot
fit well with physical reality *%sta%ta%eo$s temperat$re cha%ges do %ot occ$r a%d reactio%s
occ$r o)er a ra%ge of temperat$res at di1Kere%t rates <ore acc$rate is a ki%etic paradigm for
PC9 where reactio% rates a%d the temperat$re are al))ays cha%gi%g 6=ig 1R, right7 U%der the
e3$ili#ri$m paradigm, a cycle is deh%ed #y R temperat$res each held for a time period,
whereas the ki%etic paradigm re3$ires tra%sitio% rates a%d target temperat$res 2a#le 11 shows
typical rates a%d target temperat$res 1o$%d #y rapid&cycle PC9 6Iittwer et ai, 200,7
Paradigms are %ot right or wro%g a%d sho$ld #e @$dged #y their $sef$l%ess 2he e3$ili#&
ri$m paradigm is simple to $%dersta%d a%d le%ds itsel1 well to the e%gi%eeri%g mi%Qset 2he
ki%etic paradigm is more rele)a%t to #iochemistry, rapid PC9 a%d melti%g c$r)e a%alysis
Altho$gh most commercial *%str$me%ts still follow e3$ili#ri$m protocols, rapid proto& cols are
a %ice match for microsystems, where small )ol$mes a%d rapid PC9 are %at$ral
"igure #$% Cross+sectional diagram of a ra%id( air+controlled t$ermal cycler. An Ida$o
Tec$nology ATC 15/3 t$ermal cycler from 166/ is s$own. PCR was %ertormed in
glass ca%illaries w$ic$ were %laced in a cylindrical air c$am#er. A Central $alogen
lam% tunctioned as a $eater and an internal tan ensured e,en tem%erature
distri#ution ,,it$in t$e c$am#er. "or cooling( room tem%erature air was drawn
t$roug$ t$e c$am#er #y o%ening a door 7to% rig$t8. T$e use of air as #ot$ a $eating
and cooling medium allo,,ed for ra%id $eat transter to t$e ca%illaries( resulting in
)/+cycle am%litication times of 1/+)/ min.
6Dha%g a%d Li%g, 200-7 9apid&cycle PC9 is $sed i% real&time *%str$me%ts s$ch as the 9oche
caro$sel "igl(tCycler
0,
a%d Cepheids SmartCycler] Hther compa%ies %ow promote W=astX
protocols o% more co%)e%tio%al thermal cyclers, appare%tly i% a #id 1Mr i%depe%de%t #ra%di%g
a%d to promote reage%ts that are pres$ma#ly re3$ired =ew *%str$me%ts #ased o% microtitre
plates a%d heat #locks ca% approach rapid&cycli%g speeds a%d rapid PC9 does %ot re3$ire
special reage%ts
Real&time PCR
9eal&time PC9 %ot o%ly a$tomates #oth ampli1(catio% a%d detectio%, #$t i%tegrates them so that
they occ$r co%c$rre%tly 2ime, temperat$re a%d 1l$oresce%ce are mo%itored d$ri%g PC9 i% real&
time *%str$me%ts 2he earliest report of co%ti%$o$s mo%itori%g of PC9 a%d ac3$iri%g
E9:I;IBR<:M =I'ETIC
0 2 / S 0 10 20 R0
Time7m>n8 T>me7s8
"igure #$' E?uili#rium and @inetic %aradigms of PCR. PCR can #e descri#ed #y e?uili#rium
7lett8 or @inetic 7rig$t8 models. Bot$ %aradigms $ig$lig$t t$ree e,ents w$ic$ occur during
t$e PCR cycle 7denaturation( annealing and e-tension8. T$e more con,entional e?uili#rium
%aradigm tocuses on a s%ecitic tem%erature at ,,$ic$ a reaction occurs and its time %eriod
7e.g. e-tension at A.4c for 13 s8. T$e transitions #et,,een s%ecitic tem%eratures are
generally ignored. T$e @inetic %aradigm( $owe,er( ditters in t$at t$e tem%erature of t$e
reaction is ne,er static. &enaturation( annealing and e-tension occur o,er a range of
tem%eratures and can occur simultaneous>y. Reaction rates de%end on tem%erature #ut
are not limited to a single tem%erature. 7Re%rinted #y %ermission of Cam#ridge :ni,ersity
Press from ittwer( C.T.( Rasmussen( R.P.( and Ririe( =.M. 7.//68. Ra%id PCR and
melting analysis. In: T$e PCR Re,olution: Basic Tec$nologies and A%%lications( S.A.
Bustin( ed.8
Table #$# B%timal rates and target tem%eratures during ra%id cycle PCR under t$e @inetic
%aradigm. 7Re%rlnted #y %ermission of Cam#ridge :ni,ersity Press trom ittwer( C.T.(
Rasmussen( R.P.( and Ririe( =.M. 7.//68. Ra%id PCR and melting analysis. In: T$e PCR
Re,olution: Basic Tec$nologies and A%%lications( S.A. Bustin( ed.8
A%%roac$ rate 74cCs8
Target tem%erature 74C8
&enaturation 1/+)/ Product Tm D )
Annealing 1/+)/ Primer Tm + 3
E-tension 1+1/ 7usually .+38 53+E/ 7usually A/+A08
10 I ittwer and
1l$oresce%ce at each cycle was #y ?ig$chi et al 61,,2, 1,,R7 $tiliDi%g ethidi$m #romide, a
do$#le&stra%ded 'NA 6ds'NA7 specihc dye 2his allowed for a tr$ly homog& e%o$s or Wclosed&
t$#eX assay i% which prod$ct ampli1icatio% was com#i%ed with detectio% 2he most importa%t
applicatio% of real&time PC9 is 3$a%tihcatio% of the i%itial template, k%ow% as 3$a%titati)e
PC9 or 3PC9
9uantitati,e PCR
PC9 was desti%ed to #e a 3$a%titati)e tech%i3$e !y #oth theory a%d practice, a well&op&
timiDed PC9 do$#les the amo$%t of prod$ct each cycle for ma%y cycles Farly attempts to
har%ess the 3$a%tifyi%g power of PC9 were limited #y depe%de%ce o% e%d&poi%t a%alysis of the
prod$cts ge%erated, either #y remo)al of a% ali3$ot of the reactio% at predetermi%ed cycle
%$m#ers 6PC9 cycle titratio%C HDawa et al, 1,,07 or serial dil$tio% PC9 6!ecker et al, 1,,S7
Additio%al attempts were made to meas$re PC9 prod$cts i% the log phase of the reactio%
6Kellogg et al, 1,,0C Pa%g et al, 1,,07 or i%cl$de a competiti)e i%ter%al co%& trol i% the reactio%
6Piatak et al, 1,,R7 2hese methods were time&co%s$mi%g a%d la#o$r i%te%si)e, o1te% $si%g
agarose gels to 3$a%ti1y the amo$%t of PC9 prod$ct a%d from this determi%e a% i%itial template
co%ce%tratio%
9eal&time PC9 greatly simplihed 3$a%tihcatio% !y mo%itori%g 1l$oresce%ce o%ce each
cycle, 1l$oresce%ce as a s$rrogate of PC9 prod$ct amo$%t ca% #e plotted agai%st cycle %$m#er
No lo%ger is there a %eed to physically sample a reactio% at m$ltiple cycles or g$ess whe% PC9
is eNpo%e%tial !y ac3$iri%g data at all cycles, eNpo%e%tial data ca% #e selected i% retrospect
2he eNpo%e%tial regio% is ide%tihed #y plotti%g 1l$oresce%ce o% a log plot a%d the earliest cycle
Wsig%ihca%tly a#o)e #ackgro$%dX chose% to correlate with the i%itial template amo$%t S$ch
3$a%tihcatio% cycles 6C3s7 are $s$ally determi%ed #y either a 1l$oresce%ce threshold or #y the
maNim$m seco%d deri)ati)e *% either case, these 1ractio%al cycle %$m& #ers are i%)ersely
related to the log of the i%itial template co%ce%tratio% 2ech%ical aspects of 3PC9 a%d
per1orma%ce g$ideli%es ha)e rece%tly #ee% p$#lished 6!$sti% et al, 200,7
"luorescent indicators
9eal&time PC9 re3$ires mo%itori%g the reactio% d$ri%g amplihcatio% =l$oresce%ce is a
co%)e%ie%t method of i%terrogatio% that o%ly re3$ires a clear optical path for eNcitatio% a%d
emissio% 'o$#le&stra%Qed 'NA 6ds'NA7 dyes a%d A$oresce%tly la#elled pro#es are #oth
commo%ly $sed ds'NA dyes directly meas$re the amo$%t of do$#le&stra%ded prod$ct
prod$ced Pro#es $sed i% real&time PC9 f$%ctio% i%directly thro$gh A$oresce%ce reso& %a%ce
e%ergy tra%s1er 6=9F27 or A$oresce%ce 3$e%chi%g *%itially proposed #y 2heodor =^rster i% the
late 1,/0s 6=^rster, 1,/., 1,S07, it was %ot $%til the 1,.0s that =9F2 was applied to 'NA
6Card$llo et al, 1,..C <orriso% et al, 1,.,7 ?owe)er, real&time mo%itor& i%g with pro#es was
o%ly achie)ed se)eral years later after ds'NA dyes were esta#lished i% real&time PC9 H%e
ad)a%tage of pro#es o)er ds'NA dyes is m$ltipleNi%g #y colo$r with di1Kere%t 1l$oresce%t
dyes Ne)ertheless, this ad)a%tage comes at a cost i% i%str$me%tatio% a%d a%alysis compleNity
=$rthermore, m$ltipleN a%alysis with ds'NA dyes is possi#le #y melti%g temperat$re
separatio% of Prod$cts a%d[or pro#es
dsDNA des
ds'NA dyes are commo%place i% the molec$lar #iology la#oratory Altho$gh ethidi$m
#romide was first $sed i% real&time PC9, SY!9] 5ree% * is #y far the most commo% dye i%
real&time PC9 today *%trod$ced alo%g with the "ightCycler, it is more 1l$oresce%t tha%
ethidi$m #romide a%d is easily eNcited at the same wa)ele%gth as 1l$orescei% 6Iittwer et al,
1,,-a7 <ost real&time PC9is performed with ds'NA dyes for reaso%s of cost a%d co%&
)e%ie%ce A%y PC9 ca% #e mo%itored ))ith SY!9 5ree% * ?o))e)er, #eca$se ds'NA dyes
are ge%eric, there is a risk of %o%&specific detectio% of alter%ati)e PC9 Prod$cts 2his risk ca%
#e partly elimi%ated #y ac3$iri%g 1l$oresce%ce at a temperat$re where o%ly the desired prod$ct
is do$#le&stra%ded 6<orriso% et al, 1,,.7 <elti%g a%alysis ca% also differe%tiate #etwee%
specihc a%d %o%&specific prod$cts 69irie et al, 1,,-7
!drolsis "ro#es
*% 1,,1, ?olla%d a%d colleag$es at the Cet$s Corporatio% $sed the 0T to RT eNo%$clease acti)ity
of Taq polymerase to detect amplihcatio% Prod$cts post&PC9 6?olla%d et al, 1,,17 A%
oligo%$cleotide pro#e compleme%tary to the PC9 prod$ct was $sed with a %o%&eNte%da#le RT&
e%d a%d a radioacti)ely la#elled 0T&e%d '$ri%g amplihcatio% the polymerase degraded the
pro#e, releasi%g the radioacti)e la#el as smaller 1ragme%ts of the pro#e ?owe)er, a post&PC9
radiograph was re3$ired i% order to )is$aliDe the degraded pro#e !y replaci%g the radioacti)e
la#el with two 1l$oresce%t la#els i% a =9F2 relatio%ship, s$ccess1$l allele discrimi%atio% 6"ee
et al, 1,,R7 a%d later real&time mo%itori%g 6?eid et ai, 1,,S7 were acl(ie)ed 2hese d$al&la#elleQ
1l$oresce%t pro#es were hydrolyDed #y the 0T to RT eNo%$clease acti)ity of Taq d$ri%g PC9,
separati%g the 1l$oresce%t la#els with a loss of =9F2 to ge%erate 1l$oresce%ce Speci1(city was
e%ha%ced o)er ds'NA dyes #eca$se compleme%tatio% to three i%depe%de%t oligo%$cleotiQes
6two primers a%d o%e pro#e7 was %ecessary for pro#e hydrolysis a%d sig%al ge%eratio%
?ydrolysis pro#es 6also k%ow% #y the trademark 2a3<a%], amo%g others7 are the most
commo%ly $sed pro#es today 2heir pop$larity was ad)a%ced #y simplihed desig% 6"i)ak et al,
1,,07 a%d a stro%g commercial effort to pro)ide sy%thesis Ser)ices Sig%al ge%eratio% is
prod$ced #y pro#e hydrolysis a%d is irre)ersi#le a%d c$m$lati)e
!#ridi$ation "ro#es
*% co%trast to hydrolysis pro#es, the 1l$oresce%ce from hy#ridiDatio% pro#es is re)ersi#le a%d
depe%ds o%ly o% pro#e hy#ridiDatio% 2he 1(rst hy#ridiDatio% pro#es $sed i% real&time PC9
were d$al hy#ridiDatio% pro#es co%sisti%g of two oligo%$cleotides, o%e la#elled at the RT&e%d,
the other at the 0T&e%d 6Iittwer et al, 1,,-a7 Upo% hy#ridiDatio% to their compleme%tary
se3$e%ces a%d 1l$oresce%t eNcitatio%, =9F2 i%creases Sig%al ge%eratio% ))ith d$al
hy#ridiDatio% pro#es re3$ires a%%eali%g of fo$r oligo%$cleotides 6two primers a%d two pro#es7,
s$ggesti%g e)e% #etter specihcity tha% hydrolysis pro#es "ater, si%gle hy#ridiDatio% pro#e
desig%s ))ere de)eloped, i%cl$di%g =9F2 #et))ee% a% i%ter%ally la& #elled primer a%d a si%gle&
la#elled pro#e 6!er%ard et ai, 1,,.#C "ay a%d Iittwer, 1,,-7 a%d deoNyg$a%osi%e 3$e%chi%g of
a si%gle&la#elled pro#e 6Crockett a%d Iittwer, 20017 *% co%trast to hydrolysis pro#es that are
co%s$med d$ri%g ampli1(catio%, the 1l$oresce%ce of hy#ridiDatio% pro#es is re)ersi#le, e%a#li%g
melti%g a%alysis 2he 1irst ='A&appro)ed ge%etic tests i% the USA (%& a%d %' si%gle #ase
)aria%ts7 $sed d$al hy#ridiDatio% pro#es a%d melti%g a%alysis for ge%otypi%g
15 I ittwer and
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2he A!* --00 ))as a large
C
plate&#ased ,S&well i%str$me%t foc$sed o% hydrolysis pro#es
6!assam et al, 1,,S7 2he --00 $sed a /.. %m laser a%d fi#re opticSC i% co%trast to the light
emitti%g diodes a%d epi1l$oresce%ce optics of the "ightCycler 2hese 1(rst two real&time
*%str$me%ts are show% side&#y&side i% =ig 1S 2oday there are ma%y prod$ct o1Keri%gs i% the
are%a of real&time i%str$me%tatio% Competitio% has dri)e% dow% the costs of *%str$me%ts a%d
reage%ts =o$%datio%al de)elopme%ts i% the e)ol$tio% of PC9 a%d real&time PC9 are listed i%
2a#le 12
Current and (uture trends
!y meas$ri%g time, temperat$re, a%d 1l$oresce%ce thro$gho$t PC9, real&time R&Qime%& sio%al
spirals ca% #e ac3$ired a%d plotted 6=ig 1-7 Software o% commercial *%str$me%ts $s$ally
o%ly prese%t selected data =or eNample, 3PC9 eNperime%ts o%ly ac3$ire 1l$ores& ce%ce at o%e
temperat$re each cycle 2ypical melti%g a%alysis o%ly ac3$ires 1l$oresce%ce from o%e melti%g
c$r)e at the e%d of amplihcatio% <$ch more data is a)aila#le d$ri%g PC9, a%d it is likely that
this additio%al data will fi%d 1$rther $se i% the years to come
?omoge%eo$s mo%itori%g of PC9 is the method of choice for ge%e eNpressio% 3$a%tihcatio%
a%d closed&t$#e ge%otypi%g As a Wgold Sta%dardC it has e)ol)ed from early co%ceptio% to
prese%t&day reality =$t$re impro)eme%ts will #e t#c$sed o% red$ci%g cost a%d compleNity
6high&resol$tio% melti%g7, decreasi%g reactio% )ol$mes 6microtl$idic PC97 a%d i%creasi%g
thro$ghp$t a%d se%siti)ity 6digital PC97 2hese approaches will allow homoge%eo$s
mo%itori%g of PC9 to co%ti%$e its e)ol$tio% as a $sef$l tool for ma%y years to come
"igure #$) T$e Ida$o Tec$nology ;C .0 was t$e first commercial carousel ;ig$tCycler( tlrst
sold in 1665. GG$ile t$e %rototy%e used discrete e-citation and emission %at$s( -enon
lam% e-cltation and PMT detection( t$e commercial %roduct used e%itluorescent
interrogation of t$e ca%illary ti%( ;E&s and %$otodiodes.
./ I ittwerand
In,ention of t$e PCR
:se of a t$ermosta#le %olymerase
Ra%id cycle PCR
Hydrolysis %ro#es 7radiola#elled8
Et$idium #romide real+time PCR
Closed+tu#e allele discrimination
?PCR wit$ $ydrolysis %ro#es
SFBR green I and $y#ridiHation %ro#e real+
time PCR
Product ditterentiation #y melting
analysis
"igure #$* T$e Roc$e carousel ;ig$tCycler 7rig$t8 and t$e ABI
AA// 7left8 were t$e tirst two real+time Instruments mar@eted
worldwide. Besides t$e o#,ious ditterence in instrument siHe(
t$e #atc$ siHe 7). ,s. 658( sam%le tormat 7ca%illary ,s. %late8(
e-citation 7laser ,s. ;E&8( detection 7CC& ,s. %$otodiode8(
o%tical %at$ 7ti#re o%tic ,s. e%itluorescence8 and cycle s%eed
7ra%id+cycle ,s. Standard8 were as different as t$e grou%s t$at
created t$em. T$e ;ig$tCycler ena#led 13+)/ min PCR wit$
real+time dis%lay of tluorescence during am%li2ication( allowing
users to watc$ am%litication cur,es grow in real+time. T$e
AA// cycled more sam%les at once in %late 2ormat( #ut t$e
results could not #e o#ser,ed during am%litication and were
analysed only after com%letion of t$e run. T$e ;ig$tCycler was
introduced wit$ SFBR Ireen I and dual $y#ridiHation %ro#es
w$ile t$e AA// used $ydrolysis %ro#es. T$e carousel
;ig$tCycler #ecame a ta,ourite for ra%id de,elo%ment and
analysis ,,$ere turn+around+times were im%ortant w$ile t$e
AA// was ado%ted #y many large la#s ,,$ere Processing
con,enience and #atc$ siHe were %aramount.
Ta#le1.. Early PCR inno,ations
Inno,ation Fear Re2erence
16E.+16E3 Sai@i et al. 716E38
16EE Sai@i etal. 716EE8
166/ ittwer et al. 7166/8
1661 Holland etal. 716618
166.+166) Higuc$i etal. 7166)8
166) ;ee et al. 7166)8
1665 Heid etal. 716658
166A ittwer ef al. 7166Aa8
166A Ririe et al. 7166A8
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'igital PC9 impro)es detectio% speci1icity a%d se%siti)ity i% samples with a large #ackgro$%d of wild&type alleles
compared to )aria%t alleles a%d is the $ltimate i% allele 3$a%tihcatio% 2he red$ced cost associated with micro1l$idic
de)ices may e)e%t$ally make si%gle&step, highly parallel i%di)id$al PC9 reactio%s for digital PC9 a1Korda#le
Conclusions
PC9 has come a lo%g way i% 20 years !ased o% the f$%dame%tal properties of 'NA de%at$ratio%, hy#ridiDatio% a%d
polymerase eNte%sio%, it pro)ides a #asic tool to amplify selected 'NA 1ragme%ts for a myriad of applicatio%s 9eal&
time PC9, #y com#i%i%g ampli& hcatio% a%d detectio%, has #ecome the gold Sta%dard of %$cleic acid 3$a%tihcatio%
<elti%g a%alysis i%tegrates a% a%alysis compo%e%t witl( i%1Mrmatio% co%te%t that scales with the resol$tio% of melti%g
H)er the years PC9 has #ecome faster
C
#etter, a%d less eNpe%si)e 2he o%ly re3$ireme%ts are oligo%$cleotide primers
a%d the a#ility to temperat$re cycle, ope%& i%g $p compleN ge%etic a%alysis to all scie%tists ))ith a %eed or i%terest 2he
i%)e%tio% a%d co%ti%$ed i%%o)atio% of PC9tech%i3$es is tr$ly Wmagic i% sol$tio%
Reterences
Agarwal, K", !$chi, ?, Car$thers, <?, 5$pta, N, Khora%a, ?5, Kleppe, KC K$mar, AC Hhts$ka, F, 9a_#ha%dary, U", 8a% de
Sa%de, >?, ct a. 61,-07 2otal sy%thesis of the ge%e for a% ala%i%e tra%s1er ri#o%$cleic acid 1rom yeast Nat$re 22-, 2-&R/
!altimore, ' 61,-07 9NA&depe%de%t 'NA polymerase i% )irio%s of 9NA t$mo$r )ir$ses Nat$re 22S, 120,&1211
!assam, !>, Alle%, 2, =lood, s, Ste)e%s, >, Iyatt, 9, a%d "i)ak, K> 61,,S7 N$cleic acid se3$e%ce de& tectio% Systems+ re)ol$tio%ary
a$tomatio% for mo%itori%g a%d reporti%g PC9 prod$cts A$stralasia% !iotech%ology S, 2.0&2,/
!ecker&A%dre, <, a%d ?ahl#rock, K 61,.,7 A#sol$te m9NA 3$a%ti1katio% $si%g the polymerase chai% reactio% 6PC97 A %o)el approach
#y a PC9 aided tra%script titratio% assay 6PA22Y7 N$cleic Acids 9es (),,/R-&,//S
!ecker, A, 9eitl(, A, NapiwotDki, *., a%d Kade%#ach, ! 61,,S7 A 3$a%titati)e method of determi%i%g i%itial amo$%ts of 'NA #y
polymerase cha4% reactio% cycle titratio% $si%g digital imagi%g a%d a %o)el 'NA stai% A%al !iochem '+), 20/&20-
!eer, N9, ?i%dso%, !>, Iheeler, FK, ?all, S!, 9ose, KA, Ke%%edy, *<, a%d Colsto%, !w 6200-7 H%&chip, real&time, si%gle&copy
polymerase chai% reactio% i% picoliter droplets A%al Chem ),, ./-1&./-0
!er%ard, PS, A@ioka, 9S, K$sh%er, >p, a%d Iittwer, C2 6l,,.a7 ?omoge%eo$s m$ltipleN ge%otypi%g of hemochromatosis m$tatio%s
with fl$oresce%t hy#ridiDatio% pro#es Am> Pathol (&+, 1000&10S1 !er%ard, PS, "ay, <>, a%d Iittwer, C2 6l,,.#7 *%tegrated
amplihcatio% a%d detectio% of the CS--2 poi%t m$tatio% i% the methyle%etetrahydro1#late red$ctase ge%e #y il$oresce%ce reso%a%ce e%ergy
tra%sfer a%d pro#e melti%g c$r)es A%al !iochem 200, 101 10-
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!irch, 'F 61,,S7 Simplihed hot start PC9 Nat$re +/(, //0&//S
!rock, 2', a%d =reeDe, ? 61,S,7 T0ermus aqu1ticus ge% % a%d sp %, a %o%spor$lati%geNtreme thermo& phile > !acteriol ,/, 2.,&2,-
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6200,7 2he <*`F g$ideli%es+ mi%im$m i%1ormatio% for p$#licatio% of 3$a%titati)e real&time PC9 eNperime%ts Cli% Chem 00, S11&
S22
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A$oresce%ce reso%a%ce e%ergy tra%s1er Proc Natl Acad Sci GSA .0, .-,0&.-,/
Chie%, A, Fdgar, '!, a%d 2rela, >< 61,-S7 'eoNyri#o%$cleic acid polymerase from the eNtreme ther& mophile T0ernus 1qu1ticus.*.
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Cho$, Q.J 9$ssell, M., !irch, D.E., 9aymo%d, J., a%d !loch, w. (1992). Pre)e%tio% of pre&PC9 mis& primi%g a%d primer dimeriDatio%
impro)es low&copy&%$m#er ampli1(catio%s N$cleic Acids 9es '2, 1-1-&1-2R
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chai% reactio% A%al !iochem (/(, 1SR&1SS
Crockett, AH, a%d Iittwer, C2 620017 =l$orescei%&la#eled oligo%$cleotides for real&time PC9+ $si%g the i%here%t 3$e%chi%g of
deoNyg$a%osi%e %$cleotides A%al !iochem ',2, .,&,-
'A3$ila, 92, !echtel, ">, 8ideler, >A, Fro%,>>, 5orcDyca, p, a%d Kapla%, J.c. 61,,17 <aNimiDi%g se%siti)ity a%d specihcity of PC9
#y pre&ampli1(catio% heati%g N$cleic Acids 9es (,, R-/,
=a%C ?C, a%d `$ake, S9 6200-7 'etectio% of a%e$ploidy with digital polymerase chai% reactio% A%al Chem ),,-0-S&-0-,
=arrar, >s, 9eed, 5?, a%d Iittwer, C2 6200,7 ?igh resol$tio% melti%g c$r)e a%alysis for mo4ec$lar diag%ostics *% <olec$lar
'iag%ostics, 2%d editio%, Patri%os, 5, a%d A%sorge, w., eds 6Academic Press7, pp 22,&2/0
=i%dlay, >!, Atwood, S<, !ergmeyer, ", Chemelli,Christy, K, C$mmi%s, 2, 'o%ish, w., FkeDe, 2, =al)o,Patterso%, ', et 1. 61,,R7
.5 I ittwerand
A$tomated closed&)essel System for in vitro diag%ostics #ased o% polymerase chai% reactio% Cli% Chem R,, 1,2-&1,RR
=orster, 2 61,/.7 Ewische%molec$lare F%ergiewa%der$%g $%d =l$oresDe%D A%% Phys 2, 00&-0
=orster, 2 61,S07 'elocaliDed eNcitatio% a%d eNcitatio% tra%s1er *% <oder% `$a%t$m Chemistry, *sta%#$l "ect$res, part ***, Si%a%ogl$, o,
ed 6New York, Academic Press7, pp ,R&1R-
Po$lkes, NS, Pa%dolh de 9i%a4dis, PP, <acdo%%ell,3., Cross, NC, a%d "$DDatto, " 61,..7 Polymerase chai% reactio% a$tomated at low
cost N$cleic Acids 9es (., 0S.-&0S..
5ermer, s, a%d ?ig$chi, 9 61,,,7 Si%gle&t$#e ge%otypi%g witho$t oligo%$cleotide pro#es 5e%ome 9es ,, -2&-.
5ree%, <, 9ok$ta%da, <, =$@i%aga, K, 9ay, 9K, 9ok$ta%da, ?, a%d 5$rgo, c 619707 <echa%ism of carci%oge%esis #y 9NA
t$mor )ir$ses * A% 9NA&depe%de%t 'NA polymerase i% m$ri%e sarcoma )ir$ses Proc Natl Acad Sci GSA S-,R.0&R,R
5$%dry, CN, 8a%derstee%, >5, 9eed, 5?, Pryor, 9>, Che%, >, a%d Iittwer, C2 6200R7 Amplico% melti%g a%alysis with la#eled
primers+ a closed&t$#e method for ditKere%tiati%g homoDygotes a%d he te ro Dy go te s Cli% Chem /,, R,S&/0S
*larriso%, '>, =l$ri, K, Seiler, K, =a%, D, F1Ke%ha$ser, CS, a%d <a%D, A 61,,R7 <icromachi%i%ga mi%& iat$riDed capillary
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?eid, CA, Ste)e%s,3., "i)ak, K>, a%d Iilliams, P< 61,,S7 9eal time 3$a%titati)e PC9 5e%ome 9es S,,.S&,,/
?errma%%, <5, '$rtschi, >', Iittwer, C2, a%d 8oelkerdi%g, K.v. 6200-7 FNpa%ded i%str$me%t com& pariso% of amplico% 'NA
melti%g a%alysis for m$tatio% sca%%i%g a%d ge%otypi%g cli% Chem 0R, 10//&10/.
?ig$chi, 9, 'olli%ger, 5, Ialsh, PS, a%d 5ri1hth, 9 61,,27 Sim$lta%eo$s amplihcatio% a%d detectio% ofspecific 'NA se3$e%ces
!iotech%ology (2, /1R&/1-
* lig$chi, 9, =ockler, c, 'olli%ger, 5, a%d Iatso%, 9 61,,R7 Ki%etic PC9a%alysis+ real&time mo%itori%g of 'NA amplihcatio% reactio%s
!iotech%ology ((, 102S&10R0
?olla%d, P<, A#ramso%, 9', Iatso%, 9, a%d 5el1a%d, '? 61,,17 'etectio% of specihc polymerase chai% reactio% prod$ct #y $tiliDi%g
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&RT eNo%$clease acti)ity of 2herm$s a3$atic$s 'NA polymer& ase Proc Natl Acad Sci USA //, -2-S&-2.0
Kellogg, 'F, 9y#alki%, *, che%, s, <$khamedo)a, N, 8lasik, 2, Sie#ert, P', a%d Che%chik, A 61,,/7 2h1aStart A%ti#ody+ Whot startX
PC9 facilitated #y a %e$traliDi%g mo%oclo%al a%ti#ody directed agai%st T1q 'NA polymerase !iotech%i3$es (.,11R/&11R-
Kellogg, 'F, S%i%sky, >>, a%d Kwok, s 619907 `$a%titatio% of ?*8&1 pro)iral 'NA relati)e to cell$lar 'NA #y the polymerase
chai% reactio% A%al !iochem (/,, 202&20.
Kiss, <<, Hrtole)a&'o%%elly, ", !eer, N9, Iar%er,>, !ailey, C5, Colsto%, B.w., 9oth#erg, ><, "i%k, '9, a%d "eamo%, >?
6200.7 ?igh&thro$ghp$t 3$a%titati)e polymerase chai% reactio% i% picoliter droplets A%al Chem /2, .,-0&.,.1
Kle%o)), ?, a%d ?e%%i%gse%, * 61,-07 Selecti)e elimi%atio% of the eNo%$clease acti)ity of the deoNyri& #o%$cleic acid polymerase from
Fscherichia coli ! #y limited proteolysis Proc Natl Acad Sci USA S0, 1S.&1-0
Ko$khare)a, *, ?ao3ia%g, ?, Yee, >, Sh$m, >, Pa$l, N, ?ogre1e, 9*, a%d "e#ede), A.v. 6200.7 ?eat acti)ata#le RT&modified dN2Ps+
sy%thesis a%d applicatio% for hot start PC9 N$cleic Acids Symp Ser 6HNf7, 20,&2S0
Kwok, s, a%d ?ig$chi, 9 61,.,7 A)oidi%g false positi)es with PC9 Nat$re RR,, 2R-&2R.
"ay, <>, a%d Iittwer, C2 61,,-7 9eal&time A$oresce%ce ge%otypi%g of factor 8 "eide% d$ri%g rapid& cycle PC9 Cli% Chem -+,
''.'4''.).
"e#ede), A8, Pa$l, N, Yee,)., 2imoshch$k, 8A, Sh$m, >, <iyagi, K, Kell$m, >, ?ogrefe, 9*, a%d Eo%, 5 6200.7 ?ot start PC9 ))ith
heat&acti)ata#le primers+ a %o)el approach for impro)ed PC9 perfor& ma%ce N$cleic Acids 9es +., e 1R1
"ee, "5, Co%%ell, C9, a%d !loch, w 61,,R7 Allelic discrimi%atio% #y %ick&tra%slatio% PC9 with A$oroge%ic pro#es N$cleic Acids
9es '(, R-S1&R-SS
"iew, <, Pryor, 9, Palais, 9, <eadows, c, Frali, <, "yo%, F, a%d Iittwer, c 6200/7 5e%otypi%g of si%gle&%$cleotide polymorphisms
#y high&resol$tio% melti%g of small amplico%s Cli% Chem 00, 110S&11S/
"i)ak, K>, =lood, S>, <armaro, >, 5i$sti, w, a%d 'eetD, K 61,,07 Hligo%$cleotides with A$oresce%t dyes at opposite e%ds pro)ide a
3$e%ched pro#e System $sef$l for detecti%g PC9 prod$ct a%d %$cleic acid hy#ridiDatio% PC9 <ethods Appl -, R0-&RS2
"o, Y<, "$%, =<, Cha%, KC, 2s$i, N!, Cho%g, KC, "a$, 2K, "e$%g, 2Y, Eee, !C, Ca%tor, C9, a%d Chi$, R.w. 6200-7 'igital
PC9 for the molec$lar detectio% of fetal chromosomal a%e$ploidy Proc Natl Acad Sci :sA (2-,1R11S&1R121
"yo%, F, a%d Iitt))er, C2 6200,7 "ightCyQer tech%ology i% molec$lar diag%ostics > <ol 'iag% ((, ,R&101
<a%D, A, ?arriso%, '>, 8erpoorte, F<>, =etti%ger, J.c., Pa$l$s, A, "$di, ?, a%d Iidmer, ?< 61,,27 Pla%ar chips tech%ology for
mi%iat$riDatio% a%d i%tegratio% of separatio% tech%i3$es i%to mo%itori%g Systems+ capillary electrophoresis o% a chip > Chromatogr
0,R, 20R&20.
<o%tgomery, ., Iittwer, C2, Palais, 9, a%d Eho$, " 6200-7 Sim$lta%eo$s m$tatio% sca%%i%g a%d ge%otypi%g #y high&resol$tio% 'NA
melti%g a%alysis Nat Protoc 2, 0,&SS
<orriso%, "F, * lalder, 2C, a%d Stols, "< 61,.,7 Sol$tio%&phase detectio% of poly%$cleotides $si%g i%teracti%g A$oresce%t la#els a%d
competiti)e hy#ridiDatio% A%al !iochem (/+, 2R1&2//
<orriso%, 2!, Ieis, >>, a%d Iittwer, C2 61,,.7 `$a%tihcatio% of low&copy tra%scripts #y co%ti%$o$s SY!9 5ree% *
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<$llis, K, Paloo%a, =, Scharf, s, Saiki, 9, ?or%, 5, a%d Frlich, ? 61,.S7 Specihc e%Dymatic ampli& 1katio% of 'NA in vitro5 the
polymerase chai% reactio% Cold Spri%g ?ar# Symp ìia%t !iol 6(, 2SR&2-R
<$llis, K! 61,,17 2he polymerase chai% reactio% i% a% a%emic mode+ how to a)oid cold oligodeoNyri#o& %$clear f$sio% PC9 <ethods
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<$llis, K!, a%d =aloo%a, =A 61,.-7 Specihc sy%thesis of 'NA in vitro )ia a polymerase&catalyDed chai% reactio% <ethods F%Dymol
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NaDare%ko, *A, !hat%agar, SK, a%d ?ohma%, 9> 61,,-7 A closed t$#e format for amplihcatio% a%d detectio% of 'NA #ased o% e%ergy
tra%s1er N$cleic Acids 9es 20, 201S 2021
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i%tegratio% of PC9 amplihcatio% a%d capillary electrophoresis i% a microfa#ricated 'NA a%alysis de)ice
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repetiti)e se3$e%ces7, choice of primi%g strategy, 92 e%Dyme a%d mastermiN are all dealt with
most e1Kecti)ely o%ly a1ter sample& related re)erse tra%scriptio%, 92&PC9, PC9 a%d 3PC9
i%hi#itio% has #ee% addressed a%d[ or elimi%ated at the )ery o$tset Si%ce C
3
)al$es ca% #e
i%1l$e%ced to appear a%ywhere from . to 00 cycles solely o% acco$%t of )aryi%g degrees of
reactio% i%hi#itio%, ack%owledgi%g, addressi%g a%d com#ati%g i%hi#itio% m$st remai% foremost
i% o$r mi%ds precedi%g a%y 3PC9 e%dea)o$r
The mathematical importance of eliminating inhibition from
+PCR assays
Altho$gh thermody%amic algorithms for predicti%g )ario$s thermody%amic properties of
%$cleic acids s$ch as melti%g temperat$re 2
m
6ie as applied to primers, pro#es a%d as&
sociated amplico%s7 ha)e #ecome more sophisticated a%d #etter estimators of reality, there
?PCR In$i#ition and Am%litication of &ifficult Tem%lates I
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sample isolatio% kits 6desig%ed to remo)e a%d[or mi%imiDe i%hi#itory co%tami%a%ts i% 14%al
%$cleic acid isolates7 a%d the P&`gassay&de)elopme%t pro@ect&ma%ageme%t tool are some
direct ways to address sample&related 3PC9 i%hi#itio% 6Nola% et ai, 200SC 5all$p a%d
Ackerma%%, 200SC "aDic et al, 200-C 5all$p a%d Ackerma%%, 200.C Sow et a. 200,C Spo%seller
et al, 200,C Hli)ier et al, 200,7
The +uasi&e,ponential nature of kinetic tluorogenic +PCR effi&
ciency estimates
Ack%owledgi%g 3PC9 as a process that is still e)o4)i%g i% its descriptio%, a%d accepti%g the
idea of co%ti%$o$sly e1?cie%t eNpo%e%tial accr$al of prod$ctX #y 3PC9 to #e %either spectre
%or a#sol$te tr$th, we %o%etheless stri)e to i%terpret 3PC9 1l$oresce%ce data i% the most
respo%si#le, precise a%d mathematically mea%i%g1$l ways possi#le 2he o#ser)ed W3$asi&
eNpo%e%tiar accr$al of 1l$oresce%t sig%al i% co%c$rre%ce witl( the 3$asi&eNpo%e%tialX
acc$m$latio% of amplico% prod$ct o)er i%creasi%g 3PC9 cycles co%stit$te solidly ma%i1est,
%o%&imagi%ary phe%ome%a, e)e% tho$gh the process is %ot co%ti%$o$sly e1hcie%t
Perhaps owi%g to the fact that commercial mastermiNes ha)e #ee% pai%staki%gly opti&
miDed o)er the years #y tale%ted others to the poi%t that a%y W9$tledge[5$esci%i&likeX dis&
similarity with a co%ti%$o$sly e1hcie%t 2&to&the&tt process 6amo%g appropriately prepared a%d
dil$ted sta%dards a%d samples7 has #ee%, for the most part, masked o% acco$%t of high& ly
refi%ed prod$ct form$latio%s 6eg mastermiNes co%tai%i%g the most 3PC9&co%d$ci)e dN2P
a%d <g
2f
co%ce%tratio%s, 1l$oresce%ce sta#iliDers, 9HL, !SA, #etai%e a%d other additi)es, Taq
a%d[or 92 e%Dyme amo$%ts, optimal p? a%d io%ic stre%gth, etc7 Ie are still allo))ed,
mathematically, to treat ))ell&re%dered, %o%&i%hi#ited, highly e1?cie%t A$oro& ge%ic 3PC9
res$lts as ha)i%g ema%ated from a 2&to&the&tt&like process 6or Wh
A<
p&to&the ri process7 e)e%
tho$gl( it may occ$r, ki%etically, i% a ma%%er more mathematically aki% to the sigmoid
ampliicatio% models that 9$tledge, 5$esci%i a%d others ha)e proposed 6"i$ et al, 2002C
2ichopad et ai, 200RC 9$tledge et al, 200/, 200.C Peters et al, 200SC Ke#rea# et al, 200-C Spiess et
al, 200.C 5$esci%i et al, 200.7 24iere1ore, tr$sti%g i% the o)erall impressio% that 6gi)e% good
primer[pro#e desig%s, proper sample preparatio%, re)erse tra%scriptio% a%d PC9 sample a%d
reactio% 1orm$latio%s7, we ca% still $se a relati)e4y simplistic le%s thro$gh which to )iew
res$lts of the 3PC9 process <$ch of o$r atte%tio% sho$ld foc$s o% thi%gs that ca% help or h$rt
the acti)ity of the e%Dymes i%)ol)edC 9NA&depe%de%t 'NA& polymerase re)erse tra%scriptase
6927 e%Dyme 6eg for 14rst&stra%d, c'NA sy%thesis7 a%d thermosta#le 'NA&depe%de%t 'NA&
polymerase e%Dyme 6eg Taq for PC97
Causes of inhibition
Ihe% re)erse tra%scriptio%, 92&PC9, PC9 a%d 3PC9 reactio%s occ$r with maNimal e1hcacy
a%d speci1icity, res$lts worthy of terms s$ch as acc$racy[ precisio% a%d Wro#$st& %essX ca% #e
reaso%a#ly a%ticipated !$t, si%ce 3PC9 is ma%&made a%d relies largely o% a com#i%atio% of
e%Dymes a%d #iomolec$les that ha)e #ee% dise%gaged from their %ati)e so$rces, we m$st #e
willi%g to accept that, witho$t the ad)e%t of fi%ely t$%ed, commercially a)aila#le mastermiNes
to carry these reactio%s o$t, 3PC9 wo$ld most likely amo$%t to %othi%g more tha% a%
i%co%se3$e%tial molec$lar skirmish 2o get these processes to work, we ha)e take% 6a%d
altered7 92 e%Dymes from a)ia% a%d m$ri%e )ir$ses, Taq from Thermus aquaticus 6a%d other
#acterially deri)ed thermosta#le 'NA&depe%de%t 'NA polymerases
?PCR In$i#ition and Am%litication of &ifficult Tem%lates I 0/
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B%erator+introduced ,ariation
*% additio% to the ma%y 3$ality co%trol 1actors already disc$ssed, %o two h$ma% operators will
perform a%y o%e of the myriad steps i%)ol)ed i% eNactly the same way, so a per1$%c& tory
prelimi%ary rit$al 6postl$de to template i%tegrity a%d 3$a%tity assessme%ts7 of testi%g
eNperime%tal sample miNt$res, * s$ggest, is a practice that is $%i3$ely poised to #e a#le to shed
the most light o% each %ew 3PC9 sample System as it arises * #elie)e this sho$ld #ecome the
$%spoke% re3$isite Sta%dard acti)ity, prel$de to all 3PC9 e%dea)o$rsC the e)e%t$al, $%i)ersal
i%trod$ctory philosophy of operatio% a%d a traditio% of the trade S$ch a% i%itially re3$ired,
respo%si#le practice wo$ld seem to dema%d stat$s as a% a#so& l$te %ecessity i% all 92&PC9,
PC9 a%d 3PC9 practices 6see also the <*`F 5$ideli%es7 <oreo)er, )aria#les i%trod$ced to
each PC9 #y the h$ma% operator d$ri%g the proced$ral workflow highlight the %eed for
$%i)ersal g$ideli%es 2hose g$ideli%es wo$ld help to e%s$re some le)el of %ormalcy for a
tech%i3$e that will co%ti%$e to #e riddled with the pote%tial for ge%erati%g disastro$s,
misleadi%g, e)e% %o%se%sical o$tcomes for se)eral more years, sho$ld a sta%dardiDed i%itial
approach a%d good sample preparatio% practices %ot #ecome the immediate, $%i)ersal modus
o"erandi. <ost of the ca)eats e%co$%tered ))ith 3PC9 are th$s c$rre%tly addressed #y the <*`F
g$ideli%es, the SPU' i%l(i#itio% assay, i%hi#itory& s$#sta%ce remo)al proQ$cts o1Kered #y
compa%ies s$ch as <H !*H "a#oratories *%c a%d others, #y proper choice of assay reage%ts
a%d e%Dymes, a%d, * also co%te%d, #y $si%g the P&`gprogram 9E
T$e J2n$i#itory c$aracteristicK of a sam%le
!efore disc$ssi%g P&ì it is importa%t to 1(rst descri#e what * call the Wi%hi#itory charac&
teristicX of a sample or a gro$p of samples 6=ig 227 Hwi%g to the fact that %$cleic acids
isolated from di1Kere%t so$rces 6pla%t material, a%imal material, soil, co%tami%ated water,
#lood, faeces, etc7 #y di1Kere%t methods i%trod$ce i%to the samples differe%t amo$%ts of
co%tami%ati%g s$#sta%ces, some of wl(ich are i%hi#itory to the 92 e%Dyme or the 3PC9 phase
to diAere%t degrees, there is a clear %eed to ide%tify the dil$tio% threshold at which s$ ch
i%hi#itio% lets $p for each represe%tati)e sample or gro$p of samples A mai% feat$re of the P&
`gprogram is that this #la%ket dil$tio% for all samples is the )ery 1irst thi%g to #e ide%& tified
#efore the assay proceeds a%y 1$rther 2he Wi%hi#itory characteristicX of each similarly isolated
sample set 6from the same #iological so$rce7 is determi%ed #y $si%g a miNt$re of all similar
sample types 6eg a small portio% ofall similar&tiss$e 9NA eNtracts miNed together7 as the
serially dil$ted sample that is eNami%ed o% a 2est Plate 2he $%derlyi%g idea here is+ Wwhat
#etter a)erage represe%tati)e is there of a sample or sample set other tha% the )ery samples
themsel)esjX S$ch sample miNt$res are called WStock r i% the P&`g)er%ac$lar, a%d whe% s$ch
miNt$res are eNami%ed o)er a% 11&poi%t dil$tio% series spaced e)e%ly o% a "H5 scale 6from %o
sample dil$tio% to 1+00,000 or more7 for each target o1i%terest, the ampli14& catio% plot of C
<

)s log of dil$tio% for each target implicitly represe%ts the eNpected a)erage 3PC9 #eha)io$r of
each i%di)id$al sample & gi)e% ide%tical isolatio% methodologies for all s$ch similar samples
Fsta#lishi%g the mi%im$m dil$tio% all samples m$st $%dergo to simply a)oid the Wgro$p
i%hi#itory 01gggS0011g is somethi%g all 3PC9 assays %eed to i%corporate & %o matter how"ure
the %$cleic acid material is tho$ght to #e goi%g i%to the 3PC9 Si%ce a%ysample, %o matter
how pai%staki%gly a%d metic$lo$sly prepared, maystill har#o$r i%hi#itory pote%tial, o#ser)i%g
the eNte%t of i%hi#itory characteristic is a% a#sol$te
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( !o: and :hen do ; measure m <NA sam"les on a s"ectro"hotometer or NanoDro"j
&hould ; use <i#o=reen> ?ico=reen>
Ans:er. Accordi%g to the <*`F g$ideli%es 3$a%ti1icatio% of 9NA i% the eNtracted samples is
importa%t #eca$se it is ad)isa#le that approNimately the same amo$%ts of 9NA #e $sed ))he%
compari%g differe%t samples ?owe)er, there are se)eral 3$a%ti1icatio% proced$res i% commo%
$se, i%cl$di%g spectrophotometry 6Na%o'ropC 2hermo Scie%ti14c7, micro& 1l$idic a%alysis
6Agile%t 2ech%ologies
,
!ioa%alyDer, !io&9ad "a#oratoriesX FNperio%7, capillary gel
electrophoresis 6`_age%s ÒANcel7, or A$oresce%t dye detectio% 6Am#io%[ Applied
!iosystemsX 9i#o5ree%7 Fach method prod$ces dilKere%t res$lts, maki%g it $%& wise to try to
compare data o#tai%ed with the di1Kere%t methods 2he pre1erred method for 3$a%tifyi%g 9NA
$ses 1l$oresce%t 9NA&#i%di%g dyes 6eg 9i#o5ree%7, which are #est for detecti%g low target
co%ce%tratio%s *% a%y case, it is ad)isa#le to meas$re all samples with a si%gle method o%ly
a%d to report this i%1Mrmatio% 6!$sti% et ai, 200,7 *% additio%, al))ays meas$re the 9NA
samples post&'Nase treatme%t 6to a)oid i%cl$di%g g'NA i% the readi%gs7 $si%g a 1acsimile of
the #u@Aer the<NA is currentl in 6post&'Nase7 as the #la%ki%g 6Deroi%g7 #$ffer for the
spectrophotometer a%d[or Na%o'rop 6see eNample i% =ig 2R7 2he other co%sideratio%s here
i%)ol)e li%earity a%d limit of detectio% 2he tr$stworthy limit of detectio% is pretty m$ch the
same for #oth spectrophotometer a%d Na%o'rop H'
-S0%m
readi%gsC #etwee% 000 a%d 00S20
2his correspo%ds to a 2 to 20 %g[pl 9NA sample 6whe% meas$red at f$ll stre%gth7 ?o))e)er,
a spectrophotometer s li%ear ra%ge #egi%s to dramatically deteriorate ))ith a#sor#a%ce
readi%gs of 12 a%d higher, while the $pper limit for Na%o'rop readi%gs wo$ld #e e3$i)ale%t
to spectrophotometer readi%gs of greater tha% /0 H% a spectrophotometer, readi%gs of 9NA
isolated from samples r1ch i% 9NA 6eg li)er a%d splee%7 ca% #e dil$ted 1+100 #e1Mre
a#sor#a%ce readi%gs are take%, while samples with medi$m amo$%ts of 9NA 6s$ch as l$%g a%d
kid%ey7 ca% #e dil$ted 1+00 for a#sor#& a%ce readi%gs Samples with lower amo$%ts of 9NA
prese%t 6de%dritic cell preparatio%s,
"igure %$' T$e J#lan@ing #ufferK for nucleic acid ?uantitication. To JHeroK or J#lan@K
s%ectro%$otometers or 'ano&ro% Instruments a%%ro%riately( t$e #uffer used s$ou>d #e
identical to w$at t$e nucleic acid sam%les are in at t$e time of t$eir A.5/nmCA.E/nm
assessments. 7T$is %articu>ar custom Heroing solution is t$en diluted wit$ water to t$e same
e-tent t$at sam%les are diluted wit$ ,,ater u%on t$eir tinal s%ectro%$otometer or 'ano&ro%
a#sor#ance measurements if tinal sam%le dilutions are needed at t$is %oint( e.g. for
s%ectro%$otometer readings. 'ano&ro% measurements would generally not re?uire additional
dilution at t$is %oint.8
##% ul -uclease&free ater
#* ul #./ T0RBO !-ase Buffer
'% ul %01ml T0RBO !-ase Enzyme
Put at
'2
3c for '. minutes
#* ul T0RBO Inactiation Reagent
orte, eery #. seconds for % minutes
micro(uge for ' minutes 4 #.5... L g
#%6 ul Recoered off of top after spin
%. ul 7. u18ii R-ase inhib9tor
##'% ul -uclease&free :ater
#%6.$. ul Custom ;eroing <olut9on
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to #e co%cer%ed with the se3$e%ce&related iss$es at ha%d !eca$se there is %o $%i)ersal set of
optimal co%ditio%s for all PC9 methods, each PC9 is likely to re3$ire speci14c optimi& Datio%
of reactio% co%ditio%s per each di1Kere%t template, primer a%d[or pro#es chose% "ack of
optimiDatio% ca% res$lt i% %o%&detecta#le PC9 prod$cts or low ampli14catio% ef& ficie%cies for a
chose% template, prese%ce of %o%&specific #a%ds or prod$cts, 1Mrmatio% of primer dimers that
compete witl( a chose% template or primer set for ampli1(catio%, a%d errors i% %$cleotide
i%corporatio% 2o #e s$re, prelimi%ary in silico mi%i%g 6$se of Comp$ter programs, we#&#ased
tools a%d data#ases7 of a%y a%d all se3$e%ce i%1Mrmatio% has #ecome a ma@or aspect of moder%
92&PC9, PC9 a%d 3PC9 assay desig%s kK
IC+ric$ tem%lates
*% ge%eral, the pro#lem with 5C&rich se3$e%ces is that they ca% form ro#$st seco%dary
str$ct$res 6stem loops a%d[or hairpi% 1Mrmatio%s7 o% acco$%t ofthe triple hydroge% #o%d& i%g
capa#ility across a %$cleic acid heliN 5C&rich regio%s resist de%at$ratio% a%d pre)e%t
a%%eali%g of PC9 primers, res$lti%g i% the prod$ctio% of shorter reada#le se3$e%ces tha% other
templates as 'NA polymerases are limited or e%tirely pre)e%ted from copyi%g thro$gh the
compleN seco%dary template str$ct$res d$ri%g 92, 92&PC9, PC9 a%d 3PC9 *t is ge%erally
ad)ised to $se higher temperat$res d$ri%g re)erse tra%scriptio% to impro)e speci1kity a%d
red$ce 5C&related seco%dary str$ct$re d$ri%g first&stra%d sy%thesis a%d to employ higher
de%at$ratio% temperat$res 6eg ,.Zc as opposed to ,/ or ,0Zc7 d$ri%g the PC9 phase to allow
for more complete de%at$ratio% a%d more e1hcie%t a%d specihc primi%g a%d s$#se3$e%t
amplihcatio% ?owe)er
C
si%ce temperat$re ad_$stme%t alo%e is %ot s$1hcie%t i% ma%y sit$atio%s,
dif=ere%t additio%al strategies ca% #e employed to amplify di1hc$lt templates H%e strategy is
the s$#stit$tio% of i%osi%e 6dl2P7 for d52P i% primers a%d[or pro#es to impro)e their
per1orma%ce o% 5C&rich templates 6#y limiti%g their ow% a#ility to form hairpi% or stem&loops7
6!$sti% et al. 200,7 while a%other strategy i%)ol)es the $se of a %$m#er of additi)es which aid
i% amplifyi%g 5C&rich se3$e%ces, most of which ca$se heliN desta#iliDatio% 6Altsh$ler, 200S7
*%deed, it is most likely a partic$lar com#i%a& tio% of two or more s$ch additi)es that comprise
ma%y of the commercially a)aila#le 3PC9 We%ha%ci%g Sol$tio%sX H%e research team rece%tly
i%dicated that $si%g a com#i%atio% of three additi)es & #etai%e, dimethyl s$l1MNide, a%d -&
deaDa&2T&deoNyg$a%osi%e 6-&deaDa&2Tl d52P7 & dramatically a%d $%i)ersally impro)ed the
amplihcatio% of 'NA se3$e%ces of three disease ge%es with 5C co%te%ts ra%gi%g from S-V to
-,V 2he key co%ce%tratio%s of each of the additi)es i% this partic$lar st$dy were+ 1R <
#etai%e 6mo%o7hydrate 6Sigma7, 0V '<SH 6Sigma7, a%d 00 p< -&deaDa&2T&d52P 69oche
'iag%ostics7
2he #e%e14t of $si%g all three additi)es together is tho$ght to #e that they pre)e%t i%tra&
molec$lar sta#le stem loops i% 5C&rich templates 6d$e to stro%g 5&C pairi%g7, with each
i%di)id$al Chemical acti%g i% a slightly di1Kere%t way '<SH is tho$ght to disr$pt #asepair&
i%g, desta#iliDi%g helical 1#rmatio% a%d there#y lo))eri%g effecti)e 2
m
, while #etai%e, called a%
Wisosta#iliDi%gX age%t, e3$aliDes the co%tri#$tio% of 5&C a%d A&2 #asepairi%g to the sta#ility of
the 'NA d$pleN 6eg ca$si%g A&2 hydroge% #o%di%g to ha)e similar stre%gth as 5&C hydroge%
#o%di%g7 -&deaDa&2T&d52P
C
s$#stit$ti%g d52P
C
red$ces the stre%gth or %$m#er of hydroge%
#o%ds with compleme%tary dC2P a%d pre)e%ts formatio% of other $%desira#le Iatso%&Crick
a%d %o%&Iatso%&Crick hydroge% #o%ded #asepairi%gs *deally, each additi)e 6as a miNt$re7 ca%
#e employed at co%ce%tratio%s that do %ot i%hi#it the
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Betaine =mono>hydrate =A15A15A?rimethylglycine @ Acarbo,y&
methylB trimethylammonium> =mono>hydrate
Pote%tial #e%e1its of$se for similar facets of 92, 92&PC9, PC9 a%d[or 3PC9+ typical s$g&
gesteQ ra%ge of $se is 10m1- < *t was demo%strated 6=rackma% et al, 1,,.7 #y N<9
a%alysis that the PC9 additi)e pro)ided #y ma%y well&k%ow% compa%ies is #etai%e or #etai%e
mo%o6hydrate7, of which a 0 < sol$tio% ca% #e p$rchased from Sigma&Aldrich 6cat %o !&
0R007 Hther prod$cts tho$ght to co%sist largely of 6or most likely co%tai%i%g7 #etai%e i%cl$de
the W5C&9*C? sol$tio% e%ha%cerX from 9oche, W2a3<aster e%ha%cerX from Fppe%dor1, W5C&
meltX from Clo%tech, FP*CFN29Fs W2A`Lpedite 59FFN 9eal&2ime PC9 <aster<iNX a%d
W=ailSafe e%ha%cerX 6formerly W<asterAmp PC9 F%ha%ceme%t 2ech%ologyX7 from
FP*CFN29F !etai%e is ge%erally $sed at a fi%al co%ce%tratio% of 10&1- < *t has also #ee%
o#ser)ed that #etai%e ser)es to stre%gthe% primer&template compleNes i% A2&rich regio%s
6<elchoir et al, 1,-RC 5eAa%d, 1,.,C Smith et al, 1,,0C 9ees et al, 1,,RC 8aradara@ et al, 1,,/C
Ieisse%stei%er et a. 1,,SC !askara% et al, 1,,SC ?e%ge%, 1,,-C ?e%ke et al, 1,,-C Iilso%,
1,,-C =rackma% et al, 1,,.C Choi et al, 1,,,C Al&So$d a%d 9ndstr^m, 2000C Shammas, 2001C
!artlett a%d Stirli%g, 200RC 5r$%e%))ald, 200RC Schmerer, 200RC Ieks#erg et al, 2000C <$sso
et al, 200S7 *t sho$ld also #e me%tio%ed that it has also #ee% demo%strated that altho$gh
prod$ct yield ca% #e red$ced #y $se of #etai%e, prod$ct speci1kity is o1te% greatly impro)ed
6=rackma% et ai, 1,,.7 9E
9esearchers e)al$ati%g #etai%e ha)e fo$%d that o12 < #etai%e a%d / m< <gCl
2
worked
the #est Note that the $se of mag%esi$m is always a parameter ope% to ad_$stme%t i% e)ery
PC9 sit$atio% *% ge%eral, more mag%esi$m i%creases 6Taq) 'NA polymerase ac& ti)ity at the
eNpe%se of specificity while lower mag%esi$m ge%erally i%creases (Taq) 'NA polymerase
specihcity #$t lesse%s e%Dyme acti)ity 6S$ch helpf$l posts are commo% o% the we# these days,
a%d o%e s$ch highly i%formati)e *%teracti)e we#site is the !io2ech%i3$es <olec$lar !iology
=or$m
C
which is a scie%ce&#ased #$lleti% #oard for tech%i3$es, tips a%d 3$estio%s co%cer%i%g
molec$lar #iology, cell #iology microscopy a%d #ioi%formatics that is eNcelle%t for 92
C
92&
PC9, PC9 a%d 3PC9 tro$#leshooti%g *t ca% #e fo$%d at+ http+[[
molec$lar#iologyfor$ms#iotech%i3$escom[7
Trehalose =%&=hydro,ymethyl>&*&A'575)&tri& hydro,y&*&
=hydro,ymethyl>tetrahydropyran&%&ylB o,y&tetrahydropyran&'575)&triol
C
#%
C%%.
##
&%C
%
.> or simplyD t:o !&glucose molecules linked by an a5a&#5#&
glycosidic bond
Pote%tial #e%e1its of$se 1orsimilar facets of92, 92&PC9, PC9a%d[or 3PC9+ trehalosehas also
#ee% reported to #e eNtremely help1$l with 5C&rich templates whe% $sed at a co%ce%& tratio% of
02 < 2rehalose #elo%gs to the same class of compo$%ds to which #etai%e #e& lo%gs, k%o))%
as compati#le sol$tesX which sta#iliDe cells, cell$lar compo%e%ts a%d e%Dymes whe% eNposed
to eNtreme co%ditio%s 2rehalose is sy%thesiDed #y a )ariety of e$karyotes, co%1erri%g tolera%ce
agai%st desiccatio%, dehydratio%, temperat$re eNtremes a%d oNidatio% Additio% of trehalose
i%creases the e%Dymatic acti)ity of se)eral e$thermal e%Dymes $sed for c'NA sy%thesis or
restrictio% digestio% of 'NA 2rehalose also e%ha%ces the primi%g specihcity i% di1Kere%tial&
display 92&PC9 thro$gh high&temperat$re primi%g a%d $se of a thermally acti)ated re)erse
tra%scriptase *% additio% to the other k%ow% low&molec$lar& weight compo$%ds that e%ha%ce
the PC9 of di19c$lt templates s$ch as '<SH, other
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of&a&s$dde%, fail <y co%cl$sio% was to r$% a dil$tio% series o% the sample a%d look for the
target agai% #y 3PC9 As a res$lt, we saw that with i%creasi%g pre&3PC9 sample 9NA dil$tio%
6a1ter the most co%ce%trated samples showed %o ampli1(catio%, like the first time7, the #9S8
sig%al act$ally got progressi)ely stro%ger $%til it reached a poi%t where it #ega% to respo%d
correctly to dil$tio% 6ie higher C
3
)al$es with more sample dil$tio%7 *t was this
i%)erse[co$%ter&i%t$iti)e ma%%er i% ))hich the third, 1M$rth a%d fifth C
3
)al$es appeared that
pro)ided the cl$e as to the s$scepti#ility of 3PC9 to i%hi#itio% 6simply7 o% acco$%t of how
m$ch sample is added to the reactio% *% the dil$tio% st$dy, the 1irst two most co%ce%trated
samples 6the f$ll&stre%gth 9NA sample, a%d the 1+10&dil$ted 9NA sample7, showed %o
ampli1icatio% 6%o C
3
)al$es7, #$t the%, at a pre&3PC9 sample dil$tio% of 1+20, a C
3
of /R00
appeared, a%d at a dil$tio% of 1+R0, a C
3
of R/2, at 1+/0 a C
3
of 2-/0, a%d the fi%al tested
sample dil$tio%s of 1+.0 a%d 1+1S0 ga)e respecti)e C
3
)al$es of 2.0R a%d 2,0S 2hese
o#ser)atio%s led me to co%cl$de that a%y %$cleic acid sample s$#@ected to re)erse
tra%scriptio%, 92&PC9, PC9 or 3PC9 of a%y )ariety ca% har#o$r )aryi%g degrees of i%hi#itory
material or pote%tial 6or perhaps stim$latory material or pote%tial7C there1ore the practice of
$si%g o%e ra%domly chose% #la%ket %$cleic acid sample dil$tio% precedi%g re)erse
tra%scriptio%, 92&PC9, PC9or 3PC9 6eg 1+0 or 1+107 for all targets, is %ot aQ)is& a#le
=$rthermore, some samples ca% #e so i%hi#itory that 92 a%d PC9&#ased assays ca% ge%erate
e%tirely #elie)a#le false&%egati)e res$lts & e)e% whe% millio%s of copies of target template are
prese%t
*% additio% to the thi%gs * lear%ed from this #asic sample dil$tio% st$dy, * also recalled that
* had %ot o#ser)ed s$ch i%hi#itory phe%ome%a d$ri%g 3PC9 per1ormeQ o% p$rihed #9S8
i%oc$la a week or so earlier A1ter all, the i%oc$la was a di1Kere%tly Wp$rifiedX )iral %$cleic acid
sample, pres$ma#ly co%tai%i%g o%ly mi%imal le1t&o)er material from the o)i%e t$r#i%ate cell
c$lt$re i% ))hich the )ir$s was allowed to replicate 2hese e)e%ts immediately eNplai%ed the
ma%y other similar o#ser)atio%s * had made o)er the pre)io$s 2 years per& 1Mrmi%g 3PC9 o%
sheep l$%g total 9NA, co%c$rre%t witl( work performed #y a colleag$e o% e3$i%e de%dritic a%d
macrophage cell c$lt$re col$m%&p$rified 9NA isolates *t was the c$m$lati)e teachi%g of these
e)e%ts that @$stified the i%)estme%t of %$mero$s ho$rs 6#e& twee% 1,,- a%d 200.7 toward
#$ildi%g a% a$tomatic FNcel 200R&#ased Comp$ter program that co$ld #e $sed to 3$ickly
ide%tify the dil$tio% ra%ges withi% which %$cleic acid samples a%d sta%dards co$ld most likely
#e eNpected to #eha)e optimally d$ri%g re)erse tra%scrip& tio%, 92&PC9, PC9 a%d 3PC9 S$ch
a tool ))o$ld $ltimately f$%ctio% to help re)eal the tr$e relati)e i%itial a#$%da%ce of a%y
ge%omic or tra%scriptomic target of i%terestC gi)e% a%y tiss$e type, a%y %$cleic acid
eNtractio%[isolatio% proced$re and an human o"erator. 2his is the way that the co%cept of
Waddressi%g sample&related 92, 92&PC9, PC9 a%d 3PC9 i%hi#itio%X first prese%ted itself to me
a%d #ecame s$ch a serio$s e%dea)o$r & o%e which * co%sidered to #e ))orthy of i%&depth
atte%tio%
As a co%se3$e%ce of the time&i%te%si)e ge%esis of P&`t it has #ecome eNtremely alie% for me
to co%template 3PC9 of a%y ki%d witho$t 1irst )iewi%g it e%tirely thro$gh the le%s of the
program Creatio% of P&`>ias allowed Wi%hi#itio%X to remai%, i% my mi%d, the first thi%g to look
for, co%1ro%t a%d do away with post&haste #efore attempti%g to impro)e assay per1orma%ce i%
a%y other way *t makes %o se%se to fi%e&t$%e re)erse tra%scriptio%, 92& PC9, PC9 or 3PC9
6i% terms of addressi%g choice of 92, 1irst&stra%d primi%g, template
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SPU' assay 2he SPU' assay i%)ol)es r$%%i%g a co%trol 3PC9 $si%g a sy%thesiDed 101 #p
amplico% 6from the potato ge%ome7 i% parallel with a% ide%tical 3PC9 reactio% that co%tai%s
#oth the spiked potato amplico% 6typically 22,000 copies of it per reactio%7 a%d the #iological
sample or Sta%dard *f the sample co%tai%s a% i%hi#itor, it will take more cycles for the potato
amplico% 1l$oresce%ce sig%al to reach C
3
1% the prese%ce of i%hi#i& tio%, the C
3
will appear
later !$t, additio%ally co%1o$%di%g s$ch a%alyses is the possi#ility that i%hi#itory age%ts may
aftect the RT a%d the 0T 3PC9 assay to differe%t degrees, a#o$t which 'r Nola% has poi%ted o$t
that it is %e)er safe to ass$me that certai% i%hi#itors will always i%hi#it all 3PC9 assays to the
same degree 2here1ore, correcti%g for i%hi#itors #y 3$a%tifyi%g a re1ere%ce ge%e is really %ot a
sol$tio% Ihile the SPG' assay ca% ide%tify the prese%ce of a% i%hi#itor, the res$lts of the RT+0T
assay may also reflect the )aria#le e1Kects of a% i%hi#itor, res$lti%g i% either more 0T amplico%s
or more RT amplico%s As a s$ggested 3$ality co%trol practice, 'r Nola% has th$s recomme%ded
$si%g #oth RT+0T a%d SPU' as& says o% e)ery 9NA sample 6Nola% et ai, 200SC 5laser, 200-7
+PCR and music
As a side&%ote to some of the serio$s%ess that a#o$%ds withi% the world of tro$#leshoot& i%g
a%d properly 1Mrm$lati%g good 92, 92&PC9, PC9 a%d 3PC9 reactio%s, there arises the
e%tertai%i%g relatio%ship #etwee% the classical PC9 e3$atio% UL
n
d L
0
627eX a%d the 1Mrm$la for
1re3$e%cy 6i% ?ertD7 of m$sical %otes 6to%es o% a Sta%dard siDe, A
//0?D
pia%o7, ?D
%
d ?D
0
627r
l2

Ihat co$ld possi#ly #e lear%ed from s$ch a relatio%shipj Ple%ty *% partic$lar, whe% 3PC9
reactio%s are less tha% 100V e1hcie%t 6eg whe% eNpo%e%tial am& pli1katio% or amplico%
dou#linT falls short of a factor of 2 per cycle7, its go)er%i%g 1Mrm$la #ecomes L
C3
d L
c
6h
A<
p7
C3

C
3
)al$es ge%erated at less tha% 100V amplihcatio% e1hcie%cy, whe% compared with the same
target co%ce%tratio% di1Kere%ces represe%ted #y C
3
)al$es ge%erated at 100V e1K(cie%cy 6or h
A<
p
d 27, appear 1(irther a%d f$rther apart 2he rate of C
3
appeara%ce 6tempo7 slows dow% & yet, the
s$ccessi)e C
3
)al$es are act$ally sig%ifyi%g or playi%g the eNact same melody, o%ly o% l(igher
keys tha% the correspo%di%g 100V ef& hcie%t reactio% 2he other differe%ce here, to f$lly
accommodate the a%alogy, is that the pia%o has #ee% stretched, a%d the same UTaq4ha%dX is
merely playi%g the same t$%e o%ly o)er a lo%ger time period $si%g larger physical key
dista%ces, d$e to the stretchi%g of the key#oard 6the C
3
)al$es a%d[or pia%o keys are physically
1arther apart7 2he higher the fre3$e%cy of the %ote o% a pia%o, the less discer%i%g the h$ma%
ear is at telli%g which %ote is which 2he same thi%g applies to high C
3
)al$es ge%erated #y
3PC9 6eg C
3
)al$es a#o)e R.7 whe% P0 i%itial target template copies ge%erate i%creasi%gly
sporadic C
3
)al$es <$sic&wise a%d 3PC9&wise & the same story is #ei%g told+ ol&copy
amplihcatio%s ha)e #ee% eNperime%tally show% to eNhi#it C
3
)al$e spreads of %early siN cycles,
a%d, if we WhearX them correctly, we k%ow what melody they are si%gi%g 2hat is to say, if a
sopra%o s )i#rato is eNec$ted aro$%d the i%te%ded target to%e well e%o$gh, a well&tempered
a$die%ce k%ows she is act$ally si%gi%g a high WCX & #$t she is $si%g a ra%ge of s$rro$%di%g
to%es to physically create the vi#rato e1Kect, a%d the $se of s$ch a spread of to%es does %ot
offe%d the ear, si%ce the ear is less discer%i%g i% that 1re3$e%cy ra%ge Similarly, the sig%at$re
of si%gle&copy templates is eNperime%tally i%dicated o%ce a certai% degree of C
3
)aria%ce 6or
vi#rato7 has #ee% o#tai%ed 6eg whe% o#ser)i%g 10 replicates of si%gle&copy 3PC9 reactio%s,
C3 )al$es a%ywhere hom /R to /. ca% #e eNpected at -0V e1hcie%cy7 6"ockey et al, 1,,.7 *f
?oisson
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Al&So$d, IA, a%d 9ndstrMm, p 620007 FtKects of amplihcatio% 1acilitators o% diag%ostic PC9 i% the
Prese%ce o1#lood, feces, a%d meat > Cli% <icro#iol +/, //SR&//-0
Altsh$ler, <" 6200S7 PC9, 2ro$#leshooti%g, <"+ 2he Fsse%tial 5$ide 6Iymo%dham, GK+ Caister
AcaQemic Press7
!achma%%, !, "$ke, w, a%d ?$%sma%%, 5 61,,07 *mpro)eme%t of PC9 amplihed 'NA se3$e%ci%g with
the aid of deterge%ts N$cleic Acids 9es (/, 1R0,
!arr, P>, 2hayer, 9<, "ay#o$r%, p, Na@aria%, Seela, 9C, =, a%d 2ola%, '9 61,.S7 -&deaDa&2T&
deoNyg$a%osi%e&0 &triphosphate+ F%ha%ced 9esol$tio% i% <1R 'ideoNy Se3$e%ci%g !io2ech%i3$es
-,/2.&/R2
!artlett, ><S, a%d Stir4i%g, ' 6200R7 <ethods i% <olec$lar !iology+ PC9 Protocols 6GK+ ?$ma%a Press7
!askara%, N, Ka%dpal, 9P, !harga)a, AK, 5ly%%, <w, !ale, AC a%d Ieissma%, S< 61,,S7 U%iform
amplihcatio% of a miNt$re of deoNyri#o%$cleic acids ))ith )aryi%g 5C co%te%t 5e% 9es S, SRR&SR.
!irch, CP' 61,,,7 A %ew ge%eraliDed logistic sigmoid growth e3$atio% compareQ with the richards growth
e3$atio% A%% !ot .R, -1R&-2R
!ookstei%, 9, "ai, C&C, ?oa%g, 2, a%d "ee, I&? 61,,07 PC9&#ased detectio% of a polymorphic !am?*
site i% i%tro% 1 ofthe h$ma% reti%o#lastoma 69!7 ge%e N$cleic Acids 9es (/, 1SSS
!$sti%, SA 6200R7 A&E of `$a%titati)e PC9 6"a>olla, CA+ *%ter%atio%al U%i)ersity "i%e, !iotech%ology
Series7
!$sti%, SA 620007 9eal&time, =l$oresce%ce&#ased 3$a%titati)e PC9+ a s%apshot of c$rre%t proced$res a%d
prefere%ces FNpert 9e) i<ol 'iag% 0, /,R&/,.
!$sti%, SA 6200.7 9eal&time polymerase chai% reactio% & towards a more relia#le, acc$rate a%d rele)a%t
assay F$r Pharm 9e) S, 1,&2-
!$sti%, SA 6200.7 9eal&time 3$a%titati)e PC9 & opport$%ities a%d pit1alls F$r Pharm 9e) -, 1.&2R
!$sti%, SA, a%d Nola%, 2 6200,7 A%alysis of m9NA eNpressio% #y real&time PC9 i% 9eal&time PC9+
C$rre%t 2ech%ology a%d Applicatio%s, "oga%,)., Fdwards, K, a%d Sa$%ders, N, ed 6Nor1dlk+ Caister
Academic Press7, pp 111&1R0
!$sti%, SA, !e%es, 8, 5arso%, >A, ?ellema%s, >, ?$ggett,K$#ista, <, <$eller, 9, Nola%, 2, pfatfl, M.w.,
Shipley, 5", 8a%desompele, a%d Y8itt))er, C2 (2009). 2he <1`F 5$ideli%es+ <i%im$m *%1ormatio%
for P$#licatio% of `sa%titati)e 9eal&2ime PC9 FNperime%ts Cli% Chem 00, S11&S22
Chakra#arti, 9, a%d Sch$tt, CF 620017 2he e%ha%ceme%t of PC9 amplihcatio% #y low molec$lar weight
amides N$cleic Acids 9es 2,, 2R--&2R.1
Che)et, F, "ema1tre, 5, a%d Kati%ka, <' 61,,07 "ow co%ce%tratio%s of tetramethyl&ammo%i$m chlo& ride
i%crease yield a%d speci1kity of PC9 N$cleic Acids 9es 2R, RR/R&RR//
Choi,>&S, Kim, >&S, >oe, C&H, Kim, s, ?a, K&S, a%d Park, Y&< 61,,,7 *mpro)ed cycle se3$e%ci%go1
5C&rich 'NA template FNp <ol <ed +(, 20&2/
'a%iels, ' 6200.7 3PC9 <ethods FNpa%d rapidly+ high&thro$ghp$t plat1orms a%d real&time prohli%g
ad)a%ce discipli%e 5e% F%gi% !iotech News '/, 1,
'emeke, 2, a%d Adams, 9p 61,,27 2he etKects of pla%t polysaccharides a%d #$lKer additi)es o% PC9
!io2ech%i3$es (', RR2&RR/
'oma, <K, a%d Parker, 9 6200-7 9NA 3$ality co%tro4 i% e$karyotes Ce4l (+(, SS0&SS.
=leige, s, a%d pfaffl, <w 6200S7 9e)iew+ 9NA i%tegrity a%d the effect o% the real&time 392&PC9 perfor&
ma%ce <ol Aspects <ed 2-,12S&1R,
=rackma%, s, Ko#s, 5, Simpso%, ', a%d Storts, ' 61,,.7 !etai%e a%d '<SH+ e%ha%ci%g age%ts for PC9
Promega Notes S0,2-&R0
5all$p, ><, Kawashima, K, "$cero, 5, a%d Ackerma%%, <9 620007 New 3$ick method for isolati%g
9NA from laser capt$red cells stai%ed #y imm$%o1l$oresce%t imm$%ohistochemistryC 9NA s$ita#le for
direct $se i% 1l$oroge%ic 2a3<a% o%e&step rea4&time 92&PC9 !iol Proced H%li%e -, -0&,2
5al4$p, ><, a%d Ackerma%%, <9 6200S7 Addressi%g A$oroge%ic real&time 3PC9 i%hi#itio% $si%g the %o)e4
c$stom FLCF" file System W=oc$s=ield2&S5all$p3PC9Set&$p2ool&00r to attai% co%siste%tly high hdelity
3PC9 reactio%s !iol Proced H%li%e /, .-&100
5all$p, ><, a%d Ackerma%%, <9 6200.7 2he WP9FNcel&`g<ethodX for 3PC9 *%t > !iomed Sci /, 2-R&
2,R
5all$p, ><, Sow, =!, 8a% 5eele%, A, a%d Ackerma%%, <9 6200,7 SPG' 3PC9 Assay Co%hrms
P9FNcel&`gSoftwares A#ility to A)oid 3PC9 *%hi#itio% C$rr *ss$es <ol !iol (', 12,&1R/
5elfa%d, '? 61,.,7 Taq 'NA polymerase *% PC9 2ech%ology+ Pri%ciples a%d Applicatio%s for 'NA
Amplihcatio%, Frlich, ?A, ed 6New York+ Stockto% Press7, pp 1-&22
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Shammas, F.v. 620017 *mpro)eme%t of 3$a%titati)e PC9 reprod$ci#ility #y #etai%e as Qetermi%ed #y
fl$oresce%ce&#ased method !io2ech%i3$es R0, ,00&,0/
Smith, K2, "o%g, C<, !o))ma%, !, a%d <a%os, << 61,,07 :si%g cosol)e%ts to e%ha%ce PC9 ampli&
1katio% Ampli1icatio%s 0, 1S&1-
Sow, =!, 5all$p, ><, <eyerholD, 'K, a%d Ackerma%%, <9 6200,7 5e%e prohli%g st$dies i% the
%eo%atal o)i%e l$%g show e%ha%ci%g e1Kects of 8F5= o% the imm$%e respo%se 'e) Comp *mm$%ol
RR,-S1&--1
Sow, =!, 5all$p, ><, Sacco, 9F, a%d Ackerma%%, <9 6200,7 "aser capt$re microdissectio% re)is& ited
as a tool for tra%scriptomic a%alysis+ applicatio% of a% FNcel&#ased 3PC9 preparatio% software 6P9FNcel&
`7 *%t> !iomed Sci 0, 100&12/
Spiess, A&N, <$eller, N, a%d *)ell, 9 6200/7 2rehalose is a pote%t PC9 e%ha%cer+ lo))eri%g of d%a melti%g
temperat$re a%d thermal sta#iliDatio% of TiO@ polymerase #y the disaccharide trehalose Cli% Chem
00,120S&120,
Spiess, A&N, =eig, c, a%d 9itD, c 6200.7 ?ighly acc$rate sigmoidal htti%g of real&time PC9 data #y
i%trod$ci%ga parameter forasymmetry !<C !ioi%1#rm ,, 221&2R2 doi+ 1011.S[1/-1&2100&,&221
Spo%se4ler, !A, de <acedo, <<, Clark, SK, 5all$p, ><, a%d >o%es, 'F 6200,7 Acti)atio% of periph&
eral #lood mo%ocytes res$lts i% more ro#$st prod$ctio% of *"&10 i% %eo%atal foals compared to ad$lt
horses 8et *mm$%ol 4mm$%opathol 12-, 1S-&1-R
Sto%e, A.c. 6200/7 FNtractio% a%d Ampli1(catio% of A%cie%t 'NA *% PC9 tech%ology+ C$rre%t *%%o)atio%s,
Ieisse%stei%er, 2, 5ritl(%, ?5, a%d 5ril4(%, A<, ed 6!oca 9ato%, ="+ C9C Press7, pp 0&,
S$D$ki, 2, ?iggi%s, P>, a%d Cra))1ord, '9 620007 Co%trol selectio% for 9NA 3$a%titatio% !io2ech%i3$es
2,, RR2&RR-
S))a%go, K", ?$dlow, I9, 2imke%, <', a%d !$o%cristia%i, <9 6200-7 'e)elopme%tal )alidatio% of a
m$ltipleN 3PC9 assay for assessi%g the 3$a%tity a%d 3$ality of %$clear 'NA i% 1ore%sic samples =ore%sic
Sci *%t 1-0, R0&/0
Swille%s, s, 5otKard, >&C, <artchal, Y, de Kercho)e dXFNaerde, A, a%d ?o$s%i, ?F 6200/7 *%sta%t
e)al$atio% of the a#sol$te i%itial %$m#er of c'NA copies from a si%gle real&time PC9 c$r)e N$deic
Acids 9es R2, e0R
2helli%, o, EorDi, w, "akaye, !, 'e !orma%, !, Co$ma%s, !, ?e%%e%, 5, 5risar, 2, *go$t, A, a%d ?ei%e%,
F 61,,,7 ?o$sekeepi%g ge%es as i%ter%al sta%dards+ $se a%d limits > !iotech%ol -0, 2,1&2,0
2ichopad, A, 'ilger, <, SchwarD, 5, a%d pfafKl, <w 6200R7 Sta%dardiDed determi%atio% of real&time PC9
eukie%cy from a si%gle reactio% set&$p N$cleic Acids 9es R1, e 122 doi+1010,R[%ar[g%gl22
2ichopad, A, pfa14l, <w, a%d 'idier, A 6200R7 2iss$e&speci1k eNpressio% patter% of #o)i%e prio%+
3$a%ti1katio% $si%g real&time 92&PC9 <ol Cell Pro#es 1-, 0&10
2ichopad, A, 'idier, A, a%d pfaffl, <w 6200/7 *%hi#itio% of real&time 92&PC9 3$a%tihcatio% d$e to tiss$e
specihc co%tami%a%ts <ol Cell Pro#es (/,/0&00
:%tergasser, A, "e1e)er, s, Patty%, =, ?ellema%s, >, a%d 8a%desompele, > 6200.7 A %ew Sta%dard for 3PC9
data 9'<" F$ro !iotech Ne))s -,/0&/1
8a%desompele, >, K$#ista, <, a%d pfartl, <w 6200,7 9e1ere%ce ge%e )alidatio% software for impro)ed
%ormaliDatio% *% 9eal&time PC9+ C$rre%t 2ech%ology a%d Appl icatio%s, "oga%, Fd))ards, K, a%d
Sa$%ders, N, ed 6Nor1olk+ Caister Academic Press7, pp /-&SR
8aradara_, K, a%d Ski%%er, '< 61,,/7 'e%at$ra%ts or cosol)e%ts impro)e the specihcity of PC9 ampli&
1katio% of a 5 f C&rich 'NA $si%g ge%etically e%gi%eered 'NA polymerases 5e%e 1-2, 1 &0
88eisse%stei%er, 2, a%d "a%ch#$ry, >s 61,,S7 Strategy for co%trolli%g pre1ere%tial amplihcatio% a%d
a)oidi%g false %egati)es i% PC9typi%g !io2ech%i3$es 21, 1102&110.
Y8eks#erg, 9, ?$ghes, s, <oldo)a%, ", !assett, AS, Chow, E.w.c., a%d S3$ire, >A (2005). A method
for acc$rate detectio% of ge%omic microdeletio%s $si%g real&time 3$a%titati)e PC9 !<C 5e%omics S,
1.0&1., doi+ 1011.S[1/-1 &21S/&S&1.0
Iilso%, *5 61,,-7 <i%ire)ie))+ i%hi#itio% a%d 1acilitatio% of %$cleic acid amplihcatio% Appl F%) <icro#
SR, R-/1&R-01
Iood, I*, 5itschier, >, "asky, "A, a%d "a))%, 9< 61,.07 !ase compositio%&i%depe%de%t hy#ridiDa&
tio% i% tetramethylammo%i$m chloride+ a method for oligo%$cleotide scree%i%g of highly compleN ge%e
4i#raries Proc Natl Acad Sci USA /',10.0&10..
Yada), SP, ?a33i, 2,Ehao, L, a%d Pa%ci$, A 620027 Se3$e%ci%gi% the prese%ce o1!etai%e+ *mpro)eme%t i%
se3$e%ci%g of localiDeQ repeat se3$e%ce regio%s A!9= a#stract at http://wmv.abrf.org/Other/
A!9=<eeti%gs[A!9=2002[2002A#stractshtml 2he "er%er 9es *%stit$te, Cle)ela%d, H?
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the cross&co%tami%atio% may #e, it is still )ital to i%cl$de the N2C reactio%s i% all f$t$re
eNperime%tal r$%s to satis1y that it has %ot rec$rred A trickier pro#lem to address is whe% the
co%tami%ati%g template is #ei%g i%trod$ced at a% earlier step of the PC9 protocol a%d i% this
case co$ld ha)e a %$m#er of di1Kere%t so$rces =or eNample, a%y ge%omic 'NA prese%t i% the
sample co$ld #e the so$rce of ampli1(catio% i% a positi)e N2C reactio% a%d sho$ld #e
co%sidered especially whe% $si%g 9NA as the primary template for ampli1(catio% 6ge%omic
'NA co%tami%atio% is disc$ssed i% the s$#se3$e%t sectio% o% %o 92 Co%trols7 A%other
commo% so$rce of co%tami%atio% ca% #e from the amplico% of a pre)io$s PC9 that co%tai%
se3$e%ces to which the primers of the c$rre%t PC9 ca% a%%eal 6re1erred to as carry&o)er
co%tami%atio%7 Hpe%i%g the reactio% is %ot al))ays %ecessary i% PC9 6eg most applicatio%s
of real&time PC97, #$t i% some cases it is re3$ired for a%other dow%stream process to #e
carried o$t 6eg r$%%i%g prod$ct o% agarose gel or preparatio% for se3$e%ci%g reactio%s7 *f
care is %ot take%, this co$ld release millio%s of copies of the PC9 prod$ct o%to the la#oratory
s$r1aces a%d e3$ipme%t a%d e)e%t$ally fi%d its way #ack i%to a s$#se3$e%t eNperime%t *%
ma%y of these cases, strict la#oratory operati%g proced$res ca% #e $sed to red$ce the i%cide%ce
ofthis type of co%tami%atio% 2his ca% also #e o)ercome #y separati%g rooms for reactio% set&
$p a%d a%alysis, $si%g clea% hoods, dedicated pipettes a%d tips ca% #e employed alo%g ))ith
clea% la#oratory practices ?o))e)er, the mere fact that #illo%s of amplico%s are ge%erated i% a
PC9 mea%s that it ca% still #e )ery di1hc$lt to a)oid, especially if the same primer set is #ei%g
$sed i% ma%y reactio%s, s$ch as a la#oratory completi%g high&thro$ghp$t testi%g of samples
H%e 1vrther way to red$ce this type of co%tami%atio% is to i%cl$de deoNy$ridi%e triphosphate
6dU2P7 i% yo$r reactio% set&$p to replace 6or more o1te% miNed with7 deoNythymidi%e
triphosphate 6d22P7, there#y replaci%g thymi%e #ases with $racil Iith $racil i%corporated i%to
the res$lti%g amplico%, this prod$ct ca% #e specihcally targeted $si%g the e%Dyme $racil&'NA
glycosylase 6UN57, which li#erates the $racil resid$es from the 'NA #ack#o%e, effecti)ely
degradi%g the amplico% a%d elimi%at& i%g it as a pote%tial so$rce of amplihcatio% 6"o%go et al,
1,,07 !y $si%g a thermola#ile form of this e%Dyme, it ca% #e i%cl$ded i% a% additio%al step at
the #egi%%i%g of a PC9, to selecti)ely remo)e o%ly the co%tami%ati%g Prod$cts co%tai%i%g
$racil, a%d the% the e%Dyme is deacti)ated #e1ore amplihcatio% of the tr$e template
comme%ces H%ce agai% most of these so$rces of co%tami%atio% are deri)ed from some type of
$ser&error withi% the PC9 work&flow a%d i% ma%y cases ca% #e 1$rther a)oided #y care1$l
atte%tio% to pipetti%g a%d stricter Sta%dard operati%g proced$res
A%other reaso% for i%cl$di%g a N2C reactio% is to i%dicate the prese%ce of a%y %o%& specihc
amplihcatio% A%y sp$rio$s Prod$cts that are ge%erated d$ri%g ampli1(catio% will co%s$me the
compo%e%ts of the PC9, $ltimately red$ci%g the e1hcie%cy with which the desired prod$ct is
amplihed =$rthermore, certai% detectio% methods of PC9, s$ch as whe% $si%g a do$#le&
stra%ded 'NA #i%di%g dye i% real&time detectio%, do %ot disti%g$ish #et))ee% the %o%&specific
prod$cts a%d the target amplico%, ))hich co$ld lead to i%correct calc$latio% of the amo$%t of
target i% a sample 2he $se of a N2C is importa%t i% these sit$atio%s to i%dicate the prese%ce of
this sp$rio$s amplihcatio% a%d the% ca% #e $sed to compare to a tr$e ampli1icatio% 6s$ch as a
positi)e co%trol7, $si%g melt c$r)es a%d gel electrophoresis to disti%g$ish amplico% siDes No%&
specific ampli1katio% ca% #e i%1l$e%ceQ #y a %$m#er of factors, with the most commo% c$lprits
#ei%g poor primer desig% leadi%g to primer dimers or primi%g of other regio%s of the template
6off&targeti%g7, s$#&optimal mag%esi$m io% co%ce%tratio% a%d s$#&optimal a%%eali%g
temperat$res 62a#le R17
Signi2icance of Controls and Standard Cur,es in PCR I
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No%&competiti)e *PC methods $se a disti%ct set of primers for ampli1icatio% a%d ca% #e
$sed with either a se3$e%ce already co%tai%ed i% the template %$cleic acid 6e%doge%o$s7C s$ ch
as a re1ere%ce ge%e, or from the additio% of a spiked&i% template 6eNoge%o$s7 So$rces of
eNoge%o$s co%trol template ca% #e %at$ral or sy%thetic %$cleic acids a%d co%sideri%g that
template characteristics 6se3$e%ce, seco%dary str$ct$re, 5C co%te%t a%d le%gth7 ca% a1Kect PC9
ki%etics, the #est optio% is to choose a %at$ral co%trol template which is similar to the
eNperime%tal template 6eg a )iral 'NA of a diuere%t serotype to the test sample7 ?owe)er,
#eca$se a %at$ral co%trol template is sometimes di1hc$lt to fi%d, a%other co%)e%ie%t optio% is
to $se a sy%thetic %$cleic acidC as these ca% #e prod$ced to mimic the eNperime%tal amplico%,
a%d is a $sef$l alter%ati)e to $si%g a %at$ral template Sy%thetic 9NA Co%trols ca% #e
employed to co%trol for some of the $pstream steps i% a 92&PC9 or 392&PC9 protocol whe%
$si%g 9NA as the starti%g material for the test reactio%s =or eNample, this co%trol ca% #e
s$ppleme%ted to the test template either #efore %$cleic acid isolatio% or #e1ore c'NA sy%thesis
i% order to pro)ide i%1#rmatio% o% the per1orma%ce 3$ality of these steps '$e to the
prope%sity of 9NA molec$les i% storage to degrade at a higher rate tha% 'NA molec$les,
chemically modihed sy%thetic 9NA, which is less pro%e to degradatio%, ca% also #e employed
?owe)er this sho$ld #e $sed with caref$l co%sidera& tio%, as the mere fact that this co%trol is
more sta#le mea%s that the degradatio% rate will #e di1Kere%t to the test sample 9NA a%d th$s
ca%%ot #e $sed to i%dicate the 9NA 3$ality d$ri%g storage Commercially a)aila#le co%trol
assays a%d kits are widely a)aila#le, o1Keri%g the #e%e1(t of employi%g Co%trols ))itho$t
ard$o$s optimiDatio% a%d )alidatio%, altho$gh the se3$e%ces of these assays are o1te% kept
proprietary *% ma%y cases it may #e ad)a%& tageo$s to k%ow the se3$e%ces of the co%trol
molec$les, as this e%a#les the researcher to check for a%y sig%ihca%t homology #etwee% the
co%trol a%d test primers, as this co$ld lead to amplihcatio% of %o%&specific prod$cts
a%d[orprimer dimers
Ihere possi#le a%d appropriate, a% e%doge%o$s *PC is pre1era#le to eNoge%o$s *PC, as they
mimic the eNperime%tal co%ditio%s more closely, co%sideri%g the targets are #oth prese%t o%
the same template, the compleNity of ))hich ca% affect the performa%ce of the reactio% 2he
co%trol target m$st always #e prese%t i% the test template 6eg co%ser)ed regio%s of the lws
r9NA for a prokaryotic template7 2here1Mre, e%doge%o$s *PCs ca% o%ly #e employed i%
eNperime%ts where the template is always prese%t, th$s this approach is o%ly $sef$l for
eNperime%ts desig%ed to detect partic$lar ge%es 6eg )ir$s serotypi%g7, %ot those eNperime%ts
desig%ed to detect the prese%ce of template 6eg )ir$s detectio%7 =or the latter type of
eNperime%t, or whe% a s$ita#le e%doge%o$s target ca%%ot #e fo$%dC the% a% eNoge%o$s *PC
m$st #e $sed 2he e%doge%o$s co%trol target sho$ld prefera#ly #e prese%t i% the same copy
%$m#er as the target o1i%terest, to e%s$re that apositi)e amplihca& tio% res$lt for the co%trol
target implies that the target of i%terest sho$ld also #e detect& a#le if it is prese%t As a%
e%doge%o$s co%trol, re1ere%ce ge%es ca% #e $se1$l, as they are k%o))% to al))ays #e prese%t i%
the sample at a sta#le a#$%da%ce ?o))e)er, %o ge%e is the per1ect Who$sekeeperX, f$lfilli%g the
sta#ility characteristics i% all tiss$es a%d eNperime%tal co%ditio%s Choice of e%doge%o$s
re1ere%ces is largely depe%da%t o% the sample tiss$e or cell type, its de)elopme%tal stage, a%d
the eNperime%tal co%ditio%s employed i% the st$dy *% order to make the right choice, it is
%ecessary to test a ra%ge of ca%didate ge%es which display the desired sta#ility of eNpressio%
o% a st$dy to st$dy #asis 6Hls)ik et al, 2000C pfafKl et al, 200/C 8a%desompele et al, 20027
Signi2icance of Controls and Standard Cur,es in PCR I
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B#taining Ma-imum PCR Sensiti,ity and S%eciticity I 6#
&e2initions of sensiti,ity and s%eci2icity
!efore mo)i%g o% to the desig% a%d optimiDatio% 1$%dame%tals, some #rief deh%itio%s are i%
order A%alytical se%siti)ity for 3PC9 assays is defi%ed as the mi%im$m %$m#er of target
copies i% a sample that ca% #e detected $si%g a gi)e% assay a%d eNperime%tal co%di& tio%s
2his is typically reported as limit of detectio% 6"H'7, a%d is $s$ally eNpressed as a pro#a#ility
of detectio%, A pro#a#ility of ,0V, for eNample, mea%s that there is o%ly a 0V cha%ce of
faili%g to detect the orga%ism or target se3$e%ce at the "H' 6!$sti% et al, 200,7 PC9
se%siti)ity ca% also #e reported as the perce%tage of samples correctly ide%tit(ed =or eNample,
,.V se%siti)ity mea%s ,.V of samples were correctly ide%tihed while 2V were i%correctly
iQe%ti1ied as %egati)e
A%alytical speQAcity re1ers to the a#ility of the assay to ide%tify the desired target, a%d %ot
$%desired targets P$t a%other way, specihcity refers to the proportio% of %egati)e sam& ples
that are correctly ide%tit(ed as %egati)e, i%cl$di%g other orga%isms or target se3$e%ces 6or
a%other ge%otype7 =or eNample, i% the detectio% of i%1ectio$s orga%isms, speciAcity wo$lQ #e
eNami%ed #y testi%g whether the assay detects o%ly the desired species *f the assay pro)ides a
positi)e res$lt ))ith a %o%&targeted orga%ism, a false positi)e has occ$rred Ni%ety&siNper ce%t
speciAcity mea%s /V ofthe %egati)e samples are false positi)es
2he sit$atio% ca% #ecome more complicated ))ith 3PC9ge%e eNpressio% assays where a
false positi)e may i%dicate that a% empirically o#tai%ed di1Kere%ce i% eNpressio% is %ot act$&
ally prese%t H%e way to mi%imiDe this risk is to $se esta#lished data a%alysis criteria a%d $se
m$ltiple %ormaliDatio% ge%es 'epe%di%g o% the circ$msta%ces, higher se%siti)ity may #e
preferred e)e% at the eNpe%se of false positi)es H% the other ha%d, speciAcity m$st #e
eNceptio%ally high if the PC9 assay is diag%ostic HptimiDatio% co%ditio%s a%d assay desig%
ha)e a pro1M$%d e1Kect o% PC9 se%siti)ity a%d speciAcity
Choosing an appropriate detection <ystem
Instruments
'etectio% Systems ca% #e defi%ed as the i%str$me%t a%d 1l$oresce%t chemistry $sed to detect the
amplified 'NA *%str$me%t choice is a critical factor for good PC9 si%ce the 3$ality a%d type of
compo%e%ts determi%es the 3$ality of data that ca% #e ge%erated a%d the types of eNperime%t that
ca% #e per1Mrmed *% ge%eral, plate&#ased Systems ha)e less temperat$re precisio% tha% air&heated
Systems #$t they allow for more replicates a%d i%creased thro$ghp$t Some *%str$me%ts ha)e #etter
sig%al&to&%oise ratios thro$gh more eNpe%si)e optics, while factors s$ch as the %$m#er of discrete
1l$oresce%t cha%%els a)aila#le a%d the le)els of cross&talk #et))ee% them )ary co%sidera#ly
#etwee% diuere%t i%str$me%t types *%str$me%ts with )aria#le eNcitatio% a%d emissio% ca% assist i%
maNimiDi%g the sig%al for assays i% which the choice of A$oresce%t dyes or pro#es is i%1leNi#le 6for
eNample whe% $si%g commercial assays7 a%d software s$pport for )ario$s types of a%alyses is also
im& porta%t i% maNimiDi%g assay per1#rma%ce 2hese iss$es are treated i% more depth i% other
sectio%s of this #ook
1Mrmatio% while e)al$ati%g the 3PC9 assay with too little template will gi)e i%co%siste%t
res$lts #eca$se it is #eyo%d the "H' for the assay *f large amo$%ts of sample are re3$ired for
res$lts it has the pote%tial to i%trod$ce e3$ally large amo$%ts of i%hi#itors, pote%tially
compromisi%g the res$lts a%d loweri%g se%siti)ity
2he most commo% method for meas$ri%g %$cleic acid co%ce%tratio% is to Qetermi%e the
a#sor#a%ce at 2S0 %m 6A
2S0
7 2ypically, the 2S0[2.0 a#sor#a%ce ratio is $sed as a meas$re of
'NA or 9NA p$rity 2his is importa%t for a#sol$te or relati)e PC9 3$a%ti1katio% of samples
2he disad)a%tage of $si%g a#sor#a%ce is that a large )ol$me of a high co%ce%tra& tio% ds'NA
sol$tio% is %eeded for relia#le res$lts ?o))e)er, %ewer low )ol$me spectro& photometers 6eg
Na%odrop, 2hermo Scie%ti1ic, Na%o)$e, 5F ?ealthcare7 o%ly re3$ire 00&2 of sample to
o#tai% acc$rate res$lts Usi%g s$ch low )ol$mes re3$ires care =or eNample, i% arid
e%)iro%me%ts e)aporatio% ca% co%ce%trate %$cleic acids i% a few seco%ds leadi%g to erro%eo$s
res$lts *% adQitio%, co%tami%a%ts 1M$%Q i% %$cleic acid preparatio%s ca% a1Kect the acc$racy
ofthe res$lts
Pico5ree%g is a 1l$oresce%t %$cleic acid stai% $sed to 3$a%tify ds'NA 2he Pico5ree%
method $ses dil$tio%s of a Sta%dard ds'NA to create a Sta%dard c$r)e #ased o% 1l$ores& ce%ce
data U%k%ow% sample co%ce%tratio% is the% determi%ed #y correlati%g the 1l$ores& ce%ce to the
Sta%dard c$r)e Pico5ree% is a#le to 3$a%tify as little as 1 %g[ml of ds'NA 'iscrepa%cies
ha)e #ee% fo$%d #etwee% a#sor#a%ce a%d 1l$oresce%ce methods F)e% small amo$%ts of
cetyldimethylethylammo%i$m #romide 6C2A!7, whicl( is $sed i% ma%y 'NA eNtractio% kits,
ca% alter Pico5ree% readi%gs i% pla%t 'NA eNtracts 6?olde% et al, 200,7 2herefore, depe%di%g
o% the sample type o%e method may #e pre1era#le to the other
Additio%al 'NA from other orga%isms withi% the sample, especially whe% prese%t at l(igh
co%ce%tratio%s, ca% i%ter1ere with ampli1katio% =or eNample, a% assay desig%ed to Qe& tect a
pathoge% i% h$ma% #lood m$st #e a#le to amplify a small amo$%t ofpathoge% %$cleic acid
agai%st a large #ackgro$%d of h$ ma% ge%omic %$cleic acid 2o e)al$ate the possi#le e1Kects of
h$ma% %$cleic acids o% pathoge% detectio%, 1&S pg of h$ma% 'NA ca% #e added i%to the PC9
reactio%, a%d a%y a1Kect o% the "H' ca% #e eNami%ed *% =ig /0 two di1Ker& e%t assays are
compared for their a#ility to mai%tai% se%siti)ity at pre)io$sly Qetermi%ed "H' le)els
'i1Kere%t assays #eha)e di1Kere%tly to eNcess 'NA a%d some types of detectio% chemistry are
more ro#$st =or i%sta%ce, hydrolysis pro#es prod$ce good sig%als e)e% if s$#sta%tial amo$%ts
of $%desira#le prod$cts are prod$ced ?o))e)er, the large amo$%t of reso$rce co%s$mptio%
ofte% lowers 1l$oresce%ce or alters the growth slopes d$ri%g 3PC9
2emplate type ca% also res$lt i% differe%ces i% assay se%siti)ity *fplasmid templates are
$sed, the li%ear form of the template is typically a #etter s$#strate tha% the circ$lar form *% o$r
eNperie%ce
C
we ha)e fo$%d that for pathoge% detectio% assays, the "H' decreased from R& to ,&
fold for assays whe% li%eariDed plasmid templates were $sed ?o))e)er, for other as& says,
there was %o apprecia#le impro)eme%t i% the "H'
Aoid amplicon and enironmental contamination
!eca$se of the high se%siti)ity i%here%t to PC9 e)e% small amo$%ts of amplico% or other
e%)iro%me%tal co%tami%atio% ca% ca$se 1alse positi)es <o%itori%g for amplico% co%&
tami%atio% ca% #e performed simply #y swa##i%g areas, s$spe%di%g the swa#s i% #$1Ker, a%d
$si%g tl(is #$f=er as a template for PC9 Positi)e 3PC9 res$lts i%dicate co%tami%atio%
0AA I Iundry and Poulson
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RT+PCR B%timiHation Strategies I #.2
primer #i%di%g siteSC the #eaco% pro#e will hy#ridiDe to the template a%dC shi1t i%to a% ope%
co%hg$ratio% 2his i%creases the dista%ce #etwee% the 1l$orophore a%d the 3$e%cheKC al&
lowi%g the emitted light of the 1l$orophore to #e detected 2he occ$rre%ce of a mismatch
#etwee% a #eaco% pro#e a%d its target has a greater desta#iliDi%g e1Kect o% the d$pleN tha% the
i%trod$ctio% of a% e3$i)ale%t mismatch #etwee% the target a%d a li%ear oligo&pro#e #eca$se
the hairpi% str$ct$re pro)ides a highly sta#le alter%ate co%formatio% 2here1Mre, #eaco%s show
more speci1(city tha% the more commo% li%ear oligo&pro#es maki%g them ideal ca%didates for
detecti%g SNPs 62yagi et al, 1,,.7 <olec$lar #eaco%s ca% #e $sed i% %$mero$s applicatio%s
like m$ltipleN PC9C SNP a%alysiSC a%d detectio% of e%Dymatic clea)age 6!$sti% et al, 200/7
Sta#iliDers or Chemical cha%ges i% the pro#e 'NA #ack#o%e are $sed to keep melt tem&
perat$re re3$ireme%ts co%sta%t #$t red$ce the re3$ired le%gthC i% order i%crease #i%di%g
affi%ity a%d speci1(city of the pro#e to the amplico% FNamples for sta#iliDers are+
x <i%or groo)e&#i%ders 6<5!C Applied !iosystems7C which are added to o%e e%d of the
li%ear pro#e to i%crease the #i%di%g a1K%ity, for eNample this is do%e i% 2a3<a% Pro#es
for SNP detectio%
x "ocked %$cleic acids 6"NAC FNi3o%C see =ig 027 which are i%serted at e)ery 0th&.th
#ase positio% i% the pro#e se3$e%ce a%d are $sed for micro9NA detectio% 6FNi3o%7C SNP
a%d allelic discrimi%atio% assays A% optimiDed set of "NA pro#es is commer& cially
a)aila#le as :%i)ersal Pro#e "i#rary 69oche 'iag%ostics7 ?ere primer a%d pro#e desig%
is per1ormed a$tomatically accordi%g to the selected target a%d refere%ce ge%e se3$e%ce
a%d pre&optimiDeQ kits are $sed for m9NA 3$a%ti1katio%
x Peptide %$cleic acids 6PNA7 6Pa$laso)a a%d Pellestor, 200/7C which ha)e a peptide
se3$e%ce with a% attached 1l$oresce%ce dye PNA molec$les do %ot ha)e a %egati)ely
charged #ack#o%e 1acilitati%g stro%ger a%%eali%g to the template stra%d 6!$sti% et al, 2000C
Shipley et al. 200S7
Com%arison of detection c$emistries
Ihe% compari%g sig%als from the di1Kere%t chemistrieSC the destr$ctio% of %$clease oligo&
pro#es co%ti%$es despite a platea$ i% prod$ct acc$m$latio% whereas i%tercalati%g dye
1l$oresce%ce i% the %o template co%trol 6N2C7 ge%erally i%creases %o%&specifically d$ri%g later
cycles 2his is d$e to primer dimer which 6ca%7 acc$m$late i% late cycles 6k cycle R07
Ad_ace%t oligo&pro#e 1l$oresce%ce #egi%s to decrease as the rate of collisio% #et))ee% the
growi%g %$m#ers of compleme%tary amplico% stra%ds i%creases fa)o$ri%g the 1ormatio% of
do$#le&stra%ded 'NA o)er the hy#riQiDatio% of oligo&pro#e to its target 'NA stra%d 9eported
i% the literat$re as 2iook effectX some oligo&pro#es are co%s$med #y se3$e%ce& related
%$clease acti)ity of the $sed polymerases 6<ackay et al, 20027
All three oligo&pro#e chemistrieSC SY!9 5ree% *C 2a3<a% hydrolysis pro#es a%d ?y#Pro#es are
capa#le of detecti%g amplit(ed PC9 prod$ct with approNimately the same se%siti)ity a%d
3$a%ti1(catio% ra%ge 5e%erally the se%siti)ity of SY!9 5ree% * assays de& pe%ds stro%gly o% the
limitatio% #y $%specihc prod$ct formatio% d$e to i%e19cie%t primer desig% 2he $sage of 2a3<a%
hydrolysis pro#es or ?y#Pro#es assays offer higher specihc& ity
C
at least i% the 3$a%tihcatio% of the
ge%erated PC9prod$ct 6Iittwer et al, 1,,-7
tha%ks to its air&cooli%g a%d ce%trif$gatio% formatC a%d the *ll$mi%a Fcoy 9eal&2ime PC9
SystemC whose proprietary thermal #lock achie)es a #est&i%&class $%iformity of z 01 Zc for a
#lock&#ased i%str$me%t
2he )ariatio% i% thermal $%i1Mrmity o#ser)ed i% #lock&#ased Systems is typically d$e to
limitatio%s of the heati%g eleme%t i% co%trolli%g the thermal edge effect i%here%t i% solid #lock&
#ased desig%s Peltier eleme%ts %ormally o%ly co)er a 1ractio% of the #ottom s$r1ace of the
#lockC which ca% lead to a temperat$re gradie%t from the ce%tre to the edges of the #lock
6?errma%% et al, 200-7 Compo$%di%g tl(is edge e1Kect is the %eed to match the m$ltiple Peltier
eleme%ts %eeded to co)er the s$r1ace of a typical #lockC a task that is far hom tri)ial Some of
these effects ca% #e atte%$ated #y the $se of a passi)e re1ere%ce, s$ch as 9HLC yet $ltimately
the $ser m$st care1$lly assess the $%iformity re3$ireme%ts of his or her applicatio% a%d the
limitatio%s i%here%t i% most thermal Systems Alter%ati)ely, a $%i3$e thermal #lock desig%
s$ch as the o%e $sed #y the *ll$mi%a Fco System ca% #e $sed *t is #aseQ o% a hollow sil)er
#lock filled with a thermal co%d$cti)e fl$id that is agitated tha%ks to electromag%etic paddles
2his res$lts i% perfect thermal $%iformity across the #lock ))itho$t edge effect
S%eed
A%other aspect of the thermal System is speedC which is depe%de%t o% the rampi%g rate of the
$%it Co%)e%tio%al Peltier #locks are capa#le of heati%g a sample at l&10Zc[s
C
with cooli%g
times ge%eral4y slightly slo))er *mpro)eme%ts like sil)er a%d gold plated #locks 6*ll$mi%a
FcoyC $si%g a thermal co%d$cti)e fl$id i% its sil)er hollow #lock a%d A!* -000 =AS2C i%
com#i%atio% with the $se of =AS2 real&time PC9 chemistries7 or 2herma&!ase tech%ology 6a%
e1hcie%t heat&e3$aliDi%g layer #etwee% the heat #lock a%d the cooli%g ele& me%tC fo$%d i% the
9oche "ightCycler] /.07 ha)e i%crease sample ramp rates to 20&.ZC[s ?igh&thermal
co%d$cti)ity ceramic heati%g plates 6Cepheid SmartCycler]7 a%d resisti)e heati%g with air&
cooli%g a%d ce%tri1$gatio% 6`_age% 9otor&5e%e] `7 ca% achie)e rates as higl( as 10&20Zc[s
62a#le S17 2his tra%slates to a Sta%dard /0&cycle real&time PC9 r$% taki%g aro$%d ,0 mi%
C
with the fastest *%str$me%ts per1ormi%g the same r$% i% $%der /0 mi%
Reaction tormat
*%here%t to the thermal System is the 1ormat i% which the real&time PC9 reactio%s are e%&
closed 2he most pre)ale%t format is the ,S&well plate ))hich is a% i%d$stry Sta%dard 0 i%ch
wide #y R/ i%ch deep recta%g$lar plate that holds . rows #y 12 col$m%s of 02 ml wells
Iithi% this same lootpri%tC i%str$me%t ma%$lact$rers ha)e i%trod$ced R./ wells 6A!* -,00
=AS2 ?2C 9oche "ightCycler] /.0 a%d !io9ad C=LR./y7 a%d e)e% 10RS wells 69oche
"ightCycler] 10RS7 =or lower thro$ghp$tC ,S&well *%str$me%ts ca% hold i%di)id$& al t$#es or
t$#e&strips 9ece%tly a %$m#er of /.&well *%str$me%ts 6A!* StepH%e
1
W
81
C !io9ad
<i%iHptico%yC a%d *ll$mi%a Fcoy 9eal&2ime PC9 System7 ha)e #ee% i%trod$ced that
pro)ide a co%)e%ie%t a%d cost&effecti)e sol$tio% #et))ee% ha%dli%g i%di)id$al t$#es or t$#e&
strips a%d $si%g a portio% of a ,S&well plate per eNperime%t
Hther *%str$me%ts $seglass capillaries 69oche "ightCycler
P!k
207C Smart2$#es 6Cepheid
SmartCycler]7C or 9otor&'iscs 6ìage% 9otor&5e%e] `7 as alter%ati)es to the Sta%dard plate
format 2hese atypical 1Mrmats offer the ad)a%tage of rapid cycli%g #$t may re3$ire speci1k
additi)es to work e1Kcie%tly d$e to chemistry i%compati#ilities #etwee% )essel a%d reactio%
miN
Real+Time PCR Instrumentation: an Instrument Selection Iuide I
real&time PC9 eNperime%ts, their thermal $%its were %ot desig%ed with the eNtreme ther& mal
$%i1#rmity re3$ired for optimal real&time PC9performa%ce
Hptical Systems are made $p oftwo mai% compo%e%ts+ a% eNcitatio% ligl(t so$rce a%d a
1l$oresce%ce emissio% detector
;ig$t source
"ight so$rces ca% #e s$#gro$ped i%to two types+ %arrow or #road spectr$m
Almost all #road&spectr$m *%str$me%ts $tiliDe i%ca%desce%t t$%gste%&haloge% orNe%o%
#$l#s as their light so$rce 62a#le S17, which ge%erates higli i%te%sity light e%ergy i% the lower
part of the )isi#le ra%ge 6/-0&S/0 %m7 H%e of the mai% ad)a%tages of a #roaQ& spectr$m light
so$rce, whe% paired with a set of 1(lters, is that it pro)ides the widest choice of detecta#le
1l$orophores =or eNample, the light emitted #y the t$%gste%&haloge% lamp of the A!* -000
goes thro$gh fi)e eNcitatio% 1(lters, e%a#li%g this i%str$me%t to detect $p to fi)e di1Kere%t
1l$orophores i% the same reactio% 6k%ow% as m$ltipleNed 92&PC97 2he mai% Qisad)a%tage of
i%ca%desce%t #$l#s is that they %eed to #e replaced 6as o1te% as e)ery S mo%ths7 a%d decay o)er
time
<ost %arrow&spectr$m light so$rces 1o$%d i% real&time PC9 *%str$me%ts are light emitti%g
diodes 6"F's7 with the eNceptio% of the A!* -,00?2, which $ses a% argo%&io% laser U%like
haloge% lamps, which emit light spa%%i%g $p to 200 %m, "F's emit light of a )ery %arrow
wa)ele%gth ra%ge =or eNample a Sta%dard #l$e&gree% "F' has a peak emis& sio% of /-0 %m,
which mea%s that it ca% o%ly eNcite a limited %$m#er of 1l$orophores 6eg =A<, SY!9
697
5ree%7 2o get aro$%d this limitatio% some *%str$me%ts $se m$ltiple "F's 6!io9ad C=L,ST
<
a%d C=LR./T
<
, ìage% 9otor&5e%e
697
`, Cepheid SmartCyQer
0,
, a%d ?eliNis P*L0
1<
7, each
emitti%g light of di1Kere%t wa)ele%gths, esse%tially recreati%g the m$ltipleNi%g capa#ilities of a
#road&spectr$m light so$rce 2he mai% ad)a%tage of "F's is their lifespa%C they last $p to
/0,000 ho$rs so they %e)er %eed to #e cha%ged "F's are eNpected to o$tli)e the real&time
PC9 i%str$me%t that they are i%stalled i%
Emission detector
H%ce the %arrow& or #road&spectr$m light so$rce stim$lates the emissio% of Kl$oresce%ce #y the
1l$orophore6s7, this e%ergy %eeds to #e collected #y a 1l$oresce%ce emissio% detector 'etectors
come i% ma%y styles, i%cl$di%g CC' cameras, photodiodes, a%d photom$ltiplier t$#es 6P<2s7
2a#le S1 s$mmariDes the type of detectors fo$%d o% the di1Kere%t commer& cially a)aila#le
*%str$me%ts Fach o%e of these detectors has ad)a%tages a%d disad)a%tages+
x Photodiodes ha)e a great li%earity of o$tp$t c$rre%t as a 1ii%ctio% of light, ge%erate little
%oise, are cheap, a%d ha)e a lo%g lifetime
x P<2s are more se%siti)e a%d ha)e a 3$icker respo%se time tha% photodiodes, #$t they are
more eNpe%si)e
CC' cameras ha)e high 3$a%t$m e1hcie%cy a%d )ery li%ear o$tp$ts 2hey ca% image all wells
sim$lta%eo$sly 2hey do %ot %eed to #e mo)ed from sample to sample 6sca%& %i%g7, a%d ha)e
the ad)a%tage of %eedi%g mi%im$m mo)i%g parts leadi%g to a more ro#$st a%d cost&efficie%t
mecha%ical desig%
Real+Time PCR Instrumentation: an Instrument Selection Iuide I
Smli *OU7P2*
#7% I Hellemans and
Gandesom%ele
A,eragM e-%resston s:#Plity ,alues of rtmaining cootrol genQM
A geNorm a%alysis re3$ires a
ta#le with relati)e 3$a%tities 69ì
see #elow for calc$latio% details7
for all sample & target
com#i%atio%s as i%p$t 2his ta#le
sho$ld ha)e sample %ames as row
headers, target %ames as col$m%
headers, a% empty WA*X cell a%d
%o missi%g 9`g)al$es a%d the
f(le sho$ld #e imported i%to the
geNorm package, which ca% #e
freely dow%loaded hom
RPGQ: ACTB TBP F'HAA HMBS :/C SBHA
U%itU4ioototi
BAP
0 2 R
0200
"igure 2$' Butcome of a ge'orm analysis on a
$eterogeneous cancer #io%sy sam%le set. M ,alues 7>ine
gra%$ on to%8 for candidate reterence genes start out rat$er
$ig$ 71.)3 for B.M at t$e left8( t$en gradually dro% to,,ards
t$e more sta#ly e-%ressed genes at t$e rig$t 7TBP and
FHAS wit$ an M ,alue of a%%ro-imately /.38. T$e
analysis of V ,alues 7#ar c$art at t$e #ottom8 indicates t$at
0 reterence genes are minimally re?uired for o%timal
normaliHation in t$is $eterogeneous sam%le set 7G0C3 is
#elow /.13( indicating t$at t$ere is not need to include a
3t$ gene in t$e normaliHation tactor8. A colour ,ersion of
t$is tigure is located in t$e %late section at t$e #ac@ of t$e
6http+[[medge%$ge%t#e[o@)deso
mp[ge%orm[7, #efore start the
a%alysis Alter%ati)ely, the raw
C3 )al$es ca% #e i%p$t from yo$r
3PC9 eNperime%t i%to 3#ase
P"GS
for f$lly a$tomated a%alysis a%d
i%terpretatio% #ased o% a%
e%ha%ced geNorm algorithm *% a
geNorm a%alysis, two types of
graphs are ge%erated 2he 1irst
graph 6=ig -Ra7 ra%ks the
ca%didate re1ere%ce ge%es
accordi%g to their eNpressio%
sta#ility i% the tested samples
5e%es with )aria#le eNpressio%
6higl( geNorm < sta#ility
meas$re7 are fo$%d o% the left,
the most sta#ly eNpressed ge%es
6low < )al$e7 are located o% the
right side 2he #est two refere%ce
ge%es ca%%ot #e separated
#eca$se a geNorm sta#ility
a%alysis re3$ires at least two
ge%es 2he seco%d graph 6=ig
-R#7 ca% #e $sed to determi%e
the
?PCR &ata Analysis and Statistics I 10A
i%dicate more sta#ly eNpressed refere%ce ge%es 9efere%ce )al$es for accepta#le refere%ce
ge%e sta#ility meas$res are gi)e% i% 2a#le -1 *f all refere%ce ge%es are of poor 3$ality 6high
< a%d c) )al$es7, o%e sho$ld repeat the geNorm pilot eNperime%t o% a represe%tati)e set of
samples to fi%Q refere%ce ge%es that are s$19cie%tly sta#ly eNpressed *% sit$atio%s where o%ly
o%e o$t of three or more re1ere%ce ge%es has s$#sta%tial higher < a%d c8 )al$es tha% the
proposed refere%ce )al$es, the i%appropriate refere%ce ge%e sho$ld #e eNcl$ded from the
%ormaliDatio% proced$res As eNplai%ed i% the origi%al geNorm paper, there is a trade&
of=#etwee% cost&e1Kecti)e%ess 6fewer re1ere%ce ge%es7 a%d acc$racy 6more refere%ce ge%es7
2he program calc$lates the mi%imal %$m#er of ge%es re3$ired for relia#le %ormaliDatio%
<tatistics
*t is #eyo%d the scope of this chapter to re)iew all statistical tests to determi%e sig%i1ka%ce of a
di1Kere%ce i% ge%e eNpressio% #et))ee% 2 or more gro$ps, to ide%tify a diag%ostic or prog%ostic
9NA marker with high co%hde%ce, to fi%d correlatio%s #etwee% ge%e eNpressio% patter%s or
samples, or to ide%ti1y rele)a%t pathways or %ew sample s$#gro$ps ?owe)er, a few importa%t
poi%ts ca% #e co%sidered
=irst, it is good practice to log tra%s1orm the fi%al ge%e eNpressio% res$lts 6ie the
%ormaliDeQ relati)e 3$a%tities7C i% order to make the data distri#$tio% symmetrical 6as ge%e
eNpressio% data is o1te% log %ormally distri#$ted7 6=ig -07 2ogether with the Ce%tral limit
theorem, this allows the $se of parametric statistical tests a%d calc$latio%s that rely o% a
distri#$tio% that resem#les a %ormal distri#$tio% 6eg classic 1&test, co%hde%ce i%ter)als,
a%alysis of)aria%ce7 6<ot$lski, ? 1,,0 *%t$iti)e #iostatistics HNford+ HN1ord U%i)ersity
Press7
Seco%dly i%depe%de%t #iological replicates are re3$ired to draw mea%i%g1$l a%d relia#le
co%cl$sio%s 2he mi%im$m %$m#er of s$ch #iological replicates depe%ds o% the statistical test
a%d o% the power o%e ))a%ts to achie)e 6eg for co%hde%ce i%ter)al a%alysiSC at least three
replicates are %eeded, for a %o%&parametric paired test 6IilcoNo% sig%ed&ra%k test7, at least siN
pairs are %eeded7 *t m$st #e clear that statistics o% repeated meas$reme%ts 6eg PC9
replicates7 are a#sol$tely %o%se%se, as o%ly tech%ical )ariatio% is meas$red
2hird, the statistical test sho$ld #e selected prior to doi%g the act$al eNperime%tC ))here#y
the choice is #ased o% the 3$estio% that %eeds to #e addressed, the %$m#er of data poi%ts, a%d
the distri#$tio% of the data *f i% do$#t, a 6#io7statisticia% sho$ld #e co%s$lted 2a#le -2
pro)ides a% o)er)iew of #asic statistical tests to assess the sig%ihca%ce of Qiffere%tial ge%e
eNpressio% <ore tha% 10 years of eNperie%ce shows $s that the ma@ority o1ge%e eNpressio%
3$estio%s ca% #e addressed #ythe tests i% 2a#le -2 2hree steps %eed to #e take% to select the
proper test are 6l7 log tra%s1orm the data 6see a#o)e7, 627 e)al$ate ))hether a paired test is
%eeded a%d 6R7 assess the distri#$tio% of the pop$latio% a%d se4ect the correspo%di%g test
Pairi%g is %eeded whe% the )al$e of o%e s$#@ect i% the first gro$p is
Ta#>e A.1 Reterence ,alues for reterence gene 9C
M c)
Homogeneous sam%le set( e.g. cultures of t$e same cell ty%e(
#lood from $ealt$y indi,iduals
/.3 .3T
Heterogeneous sam%le set( e.g. com#ination of different cell
ty%es( cancer #io%sies
1
3/T
T$e MI9E Iuidelines :ncloa@ed I 13)
is o%ly o%e optio% a%d those prod$cts ha)e #ee% refere%ced A%other co%sideratio% i% this
decisio% is the o#ser)atio% that ))hile the 1olks i% la#oratory A will ha)e great s$ccess with
prod$ct L o)er Y, those i% la#oratory ! dow% the hall will swear that prod$ct Y is m$ch #etter
tha% prod$ct L 2his ill$strates what is most likely the most $%doc$me%ted part of a%y
research pro@ect, the effect of eNperie%ce a%d the 3$ality of the perso%%el i%)ol)ed o% the
o$tcome of the eNperime%t Si%ce this factor ca% %ot #e 3$a%tihed i% a%y relia#le way
C
we ha)e
to depe%d $po% the parameters o$tli%ed i% the <*`F checklist as a meas$re of their s$ccess
U%fort$%ately there are ma%y other eNamples of 3PC9 data i% the literat$re that ha)e #ee%
improperly or i%ade3$ately reported
2he 1ollo))i%g disc$ssio% ))ill co)er the mai% topics of the <*`F checklist i% order
62a#le .17 ?ope1$lly, my comme%tary will help flesh o$t what we 6the <*`F a$thors7 had i%
mi%d whe% these items were p$t o% the list
E,perimental design
2he most importa%t compo%e%t of a%y eNperime%t is i% the pla%%i%g "ike a chess match,
looki%g se)eral mo)es ahead prior to #egi%%i%g ca% sa)e a lot of time, e%ergy a%d pote%tial
heartache dow% the road H%e of the most importa%t co%sideratio%s is i% determi%i%g how
ma%y mem#ers will #e re3$ired i% each eNperime%tal gro$p to achie)e statistical sig%i1(ca%ce
compared to o%e or more co%trol gro$ps 2his is determi%ed #y performi%g a power a%alysis
#ased o% prelimi%ary eNperime%tal data from a smaller s$#set of samples *t is importa%t to
defi%e what co%stit$tes a% eNperime%tal a%d co%tro4 gro$p, the %$m#er 6N7 i% each gro$p a%d a
clear represe%tatio% of the data from each gro$p
As a Core "a#oratory director, * i%sist that p$#licatio%s i% wl(ich the data prese%ted are
ge%erated withi% o$r facility me%tio% o$r i%)ol)eme%t i% the ack%owledgeme%ts or methods
sectio% of the paper 2he Wcoi% of the realmX for Core "a#s is pro)idi%g data for i%)estigators
that lead to p$#licatio%s a%d gra%ts
<ample preparation
2he term WsampleX i% a%y eNperime%t ca% ha)e a large ra%ge of possi#le deh%itio%s 2herefore, it is
critical that the a$thor defi%e what a sample is for each of their eNperime%ts 2his will i%cl$de a #rief
disc$ssio% of the origi% of the sample 6eg tiss$e c$lt$re, a%imal tiss$e, ==PF materialC laser
capt$re, #lood, faeces, pla%t, micro#e7C how it was o#tai%ed 6eg cell lysis, #iopsy7C how it was
ha%dled 6eg 1lash froDe%, 9NAlater, homoge%iDed immediately, hNatio%7C a%d how it was stored
prior to a%alysis 6eg temperat$re a%d time prior to %$cleic acid isolatio%7 2he amo$%t of sample
collected ca% also #e critical whe% e)al$ati%g the 9NA or 'NA isolatio% proced$re a%d sho$ld #e
reported 2he collectio% of samples is arg$a#ly the most importa%t phase ofthe eNperime%t *fthe
%$cleic acids are %ot sta#iliDed s$1hcie%tly at this step, there is %othi%g yo$ ca% do later o% to recti1y
the sit$atio% *t has #ee% my o#ser)atio% o)er the years that it is importa%t for i%)estigators who
ha)e little eNperie%ce with the collectio%, isolatio% a%d p$rihcatio% of%$cleic acids to practice all
phases of this proced$re prior to i%itiati%g what may #e a )ery costly set of eNperime%ts a%d e%s$re
that they are tech%ically $p to the challe%ge 2his is also tr$e for perso%%el who ha)e a lot of
eNperie%ce #$t are o#tai%i%g their material from a %ew so$rce Ne)er ass$me tliat past methods ))ill
work with the %ew material
$ i i i i i mmi
c&'A
"igure h$#2 lllustration of stem+loo% Rt+?PCR met$od. Ta?Man+#ased real+time ?uanti2ication of
miR'As in,ol,es two ste%s( stem+loo% RT and real+time PCR. stem+loo% RT %rimers #ind to t$e )*
end of t$e targeted miR'A t$roug$ annealing and are re,erse transcri#ed to ma@e tirst+strand c&'A
w$ic$ includes t$e miR'A and re,erse %rimer #inding site. T$e %ur%ose of t$e tailed forward %rimer
is to increase t$e melting tem%erature if needed( de%ending on t$e se?uence com%osition of t$e
miR'A.
reactio%s are i%c$#ated i% a R./&well plate at ,0Zc for 10 mi%, followeQ #y /0 cycles of ,0Zc for 10
s a%d S0Zc for 1 mi%
Ad,antages
A primary aQ)a%tage of 2a3<a% assays is their high degree of specificity a%d the fact that they
g$ara%tee the a#ility to 3$a%ti1y o%ly the mat$re mi9NAs with %o cross&reacti)ity to prec$rsors
2hey also pro)ide si%gle&#ase discrimi%atio% of closely related family mem#ers a%d a high le)el of
se%siti)ity, re3$iri%g o%ly 1&10 %g of total 9NA or e3$i)ale%tC which with the $se of a pre&
amplificatio% protocol, will e%a#le 3$a%titatio% of mi9NAs hom si%gle cells A%other #e%e1it is that
they are simple, scala#le a%d compati#le with the Cell& to&C2
1<
kit 6Applied !iosystems7 for direct
3$a%titatio% of mi9NAs hom cell lysates
Ta?ManU microR'A arrays
2a3<a%] <icro9NA Arrays pro)ide all the ad)a%tages of 2a3<a%] <icro9NA Assays i% a
co%)e%ie%t, pre&co%fig$red micro 1l$idic card mi%imiDi%g eNperime%tal )aria#ility a%d ef& fort
re3$ired to r$% R./ 2a3<a%] <icro9NA Assays i% parallel <egapleNT
<
92 Primers are highly
m$ltipleNed 92 primers desig%ed to co%)ert $p to R./ mi9NAs a%d co%trol ge%es to c'NA prior to
real&time a%alysis a%d simplify the 2a3<a%] <icro9NA Array whe% se%siti)ity is of the $tmost
importa%ce a%d[or 9NA sample is limiti%g, <egapleN
*<
PreAmp Primers pro)ide a% optio%al
preampli1(catio% step 2a3<a%] <icro9NA Arrays )20 a%d <egapleN
{<
Primer Pools e%a#le a
comprehe%si)e eNpressio% profile co%siste%t with Sa%ger mi9!ase )io withi% 0 ho$rs, pro)idi%g the
ideal mi9NA pro1ili%g sol$tio%
PCR A%%lications for E%igenetics Researc$ I 3A/
Real&time
Ta?Man %rotoe %nmor
Fou
$a,e
eit$er
reac$e
d a
%age
Fou
$a,e
eit$er
reac$e
d a
%age
Fou
$a,e
eit$er
reac$e
d a
%age
Fou
$a,e
eit$er
reac$e
d a
%age
9ey%a$d, c, !r$%o, c, !o$lla%ger, p, 5ra%ge, >, !ar#esti, s, a%d Ni)elea$, A 61,,27 <o%itori%g of $ri%ary
eNcretio% of modified %$cleosides i% ca%cer patie%ts $si%g a set of siN mo%oclo%al a%ti#odies Ca%cer "ett
.(, 200&2S2
9odrig$eDC A, 8igorito, F, Clare, s, Iarre%, <8, Co$ttet, p, Soo%d, '9, )a% 'o%ge%, s, 5rocock, 9>C
'as, PP, <iskaC FA, eta. 6200-7 9e3$ireme%t of #ic[micro9NA&100 for %ormal imm$%e 1$%ctio%
Scie%ce +(.,S0.&S11
9$)k$%, 5, 88ightma%, !C a%d ?a, * 6200/7 2he 20 years it took to recog%iDe the importa%ce of ti%y
9NAs Cell (., S,R&,SC p ,2 followi%g S,S
Schilli%g, F, a%d 9ehli, < 6200-7 5lo#al, comparati)e a%alysis of tiss$e&specihc promoter Cp5 methyla&
tio% 5e%omics ,2, R1/&R2R
Schmidl c, Kl$g, <, !oeld, 2> A%dreese% 9, ?ouma%%, 9, Fdi%ger, <, a%d 9ehli, < 6200,7 "i%eage&
specihc 'NA methylatio% i% 2 cells correlates with histo%e methylatio% a%d e%ha%cer acti)ity 5e%ome
9es (,, 11S0&11-/
Shar#ati&2ehra%i, s, K$tD&"ohro1f, !, !erg#a$er, 9, Schol)e%, >, a%d Fi%spa%ier, 9 6200.7 mi9&`@ a %o)el
3$a%titati)e 92&PC9 approach for the eNpressio% prohli%g of small 9NA molec$les s$ch as mi9NAs i% a
compleN sample !<C <ol !iol ,, R/
She%, ", 5o$, Y, Che%, L, Ahmed, s, a%d *ssa, >9 6200-7 HptimiDi%g a%%eali%g temperat$re o)ercomes
#ias i% #is$lhte PC9 methylatio% a%alysis !io2ech%i3$es /2+/.&02
Shi, 9, a%d Chia%g,8" 620007 =acile mea%s for 3$a%ti1yi%g micro9NA eNpressio% #y real&time PC9
!io2ech%i3$es +,, 01,&020
Strahl, !', a%d A4lis, C' 620007 2he la%g$age of co)a4e%t histo%e modihcatio%s Nat$re -2+, /1&/0
Swift&Sca%la%, 2, !lack1#rd, A, Arga%i, p, S$k$mar, s, a%d =ackler, <> 6200S7 2))o&color 3$a%titati)e
m$ltipleN methylatio%&specihc PC9 !io2ech%i3$es -2, 210&21,
2a1t, 9>, Kapla%, C', Simo%s, c, a%d <attick, >s 6200,7 F)ol$tio%, #ioge%esis a%d U$%ctio% of promoter&
associated 9NAs Cell cycle A$g 2+.6107
2ari3, <, a%d PasDkowski, > 6200/7 'NAa%d histo%e methylatio% i% pla%ts 2re%ds 5e%et '2, 2//&201
2a)aDoie, S9, Alarco%, c, Hskarsso%, 2, Pad$a, ', Ia%g, !os, P', 5erald, I", <assag$e,> 6200.7
F%doge%o$s h$ma% micro9NAs that s$ppress #reast ca%cer metastasis Nat$re /0(, 1/-&102
2homassi%, ?, Kress, c, a%d 5ra%ge, 2 6200/7 <ethyl`$a%t+ a se%siti)e method for 3$a%tifyi%g meth&
ylatio% ofspecific cytosi%es withi% the ge%ome NA9 +', elS.+
2reisma%, 9 61,,07 >o$r%ey to the s$r1ace of the cell+ =os reg$latio% a%d the S9F F<!H > (-, /,00&/,1R
2ri#o$let, 9, <ari, !, "i%, Y", Cha#le&!essia, c, !e%%asser, Y, "e#riga%d, K, Cardi%a$d, !, <a$ri%, 2,
!ar#ry, p, !aillat, 8, eta. 6200-7 S$ppressio% of micro9NA&sile%ci%g pathway #y ?18&1 d$ri%g)ir$s
replicatio% Scie%ce Ri0, 10-,&10.2
2$r%er, !< 620027 Cell$lar memory a%d the histo%e code Cell (((, 2.0&2,1
8oss, KH, 9oos, KP, No%ay, 9", a%d 'o)ichi, N> 61,,.7 Com#ati%g PC9 #ias i% #is$lhte&#ased cyto&
si%e methylatio% a%alysis #etai%e&modihed cytosi%e deami%atio% PC9 A%al Chem )2, R.1.&R.2R
Ie#er, <, 'a)ies, >>, Iittig, ', Hakeley, F>, ?aase, <, "am, I", a%d Schv#eler, ' 620007
Chromosome&wide a%d promoter&specihc a%alyses ide%ti1y sites of ditKere%tial 'NA methylatio% i%
%ormal a%d tra%s1ormed h$ma% cells Nat 5e%et +), .0R&.S2
Iei%ma%%, AS, a%d =ar%ham, P> 620027 *de%tihcatio% of $%k%o))% target ge%es of h$ma% tra%scriptio%
1actors $si%g chromati% imm$%oprecipitatio% <ethods 2S, R-&/-
Ie l l s , a % d =ar%ham, P> 620027 CharacteriDi%g tra%scriptio% 1actor #i%di%g sites $si%g 1ormaldehyde
cross&li%ki%g a%d imm$%oprecipitatio% <ethods, (,/.&0S
Io@dacD, 2K, a%d *4a%se%, "" 6200S7 9e)ersal of PC9 #ias for impro)ed se%siti)ity of the 'NA meth&
ylatio% melti%gc$r)e assay !io2ech%i3$es -(, 2-/&2-.
Lio%g, D, a%d "aird, pw 61,,-7 CH!9A+ a se%siti)e a%d 3$a%titati)e 'NA methylatio% assay N$cleic
Acids 9es 20, 20R2&20R/
Yeg%as$#rama%ia%, s, "i%, L, ?atK%er, <C, 'e<arDo, A<, a%d Nelso%, I5 6200S7 Com#i%atio% of
methylated&'NA precipitatio% a%d methylatio%&se%siti)e restrictio% e%Dymes 6CH<PA9F&<S7 for the
rapid, se%siti)e a%d 3$a%titati)e detectio% of 'NA methylatio% N$cleic Acids 9es R/, e 1,
Y$, S", Che%, ?Y, Cha%g, 5C, Che%, CY, Che%, ?w, Si%gh, s, Che%g, C", Y$, C>, "ee, YC, Che%,
?S, et 1i 6200.7 <icro9NA sig%at$re predicts s$r)i)al a%d relapse i% l$%g ca%cer Ca%cer Cell 1R, /.&0-
PCR A%%lications for E%igenetics Researc$ I
Fou
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eit$er
reac$e
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%age
Fou
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eit$er
reac$e
d a
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reac$e
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Hig$+Resolution Melting Analysis I %.'
a%alyse SNPs withi% the amplico%s 65$%dry et a. 200R7 ?owe)er, as with other si%gle pro#e
ge%otypi%g methods, a specihc la#elled oligo%$cleotide was re3$ired for each reac& tio%
6$%less $si%g a ge%eric i%dicator oligo%$cleotide homologo$s to a 0T tail of the assay primer
65$%dry et al. 200R77
?9<A, as it is commo%ly $sed toQay, is #ased o% mo%itori%g the melti%g of ))hole
PC9amplico%s 6amplico% ?9<A7 2his method was e%a#led #ythree de)elopme%ts 2he first
was the i%trod$ctio% of Wsat$rati%gX dyes & dyes that ca% #e $sed at high e%o$gh co%&
ce%tratio%s that heterod$pleNes are detected '$e to its i%l(i#itory effects o% PC9, SY!9
5ree% * is $sed at low co%ce%tratio%s a%d its tra%slocatio% d$ri%g melti%g c$r)e ac3$isitio%
ca% ca$se prod$ct ide%tihcatio% iss$es 65iglio et ai, 200RC <o%is et ai, 20007 2he first
sat$rati%g dye i%trod$ced was "C 5ree% * a%d was descri#ed together witl( the 1(rst high&
resol$tio% melti%g i%str$me%t, ?9&1 6#oth from *daho 2ech%ology *%c7 <a%y ?9<A
applicatio%s were i%itially de)eloped $si%g this dye a%d i%str$me%t, alo%g with software
methods a%d %ormaliDatio% steps for a%alysis 6Iittwer et al, 200R7
!yes for CR?A
Si%ce the i%trod$ctio% of "C 5ree% *, "C 5ree% P"US 6idaho 2ech%ology *%c7 was i%&
trod$ced as a #righter dye #etter adapted to microtiter plates as opposed to capillaries A
%$m#er of other dyes ha)e si%ce #ee% reported for $se as dyes i% ?9<A 2hese i%cl$de
SY20, a%d SY!9 5ree%F9 6%ot to #e co%1$sed with SY!9 5ree% (7 from *%)itroge% 6<o%is
et al, 2000C Kryp$y et al, 200SC Iolff et al, 200.7, F)a5ree% from !ioti$m 6<ao et al, 200-7,
Chromo1y a%d !F!H from 2A2AA !ioce%ter 6<ader et al. 200.7 a%d 9esolight from 9oche
Applied Scie%ce 6SchvtD a%d )o% Ahse%, 200,7 2he cost per reactio%, optimal eNcitatio%
))a)ele%gths, a%d performa%ce of these dyes )aries co%sidera#ly Some are o%ly a)aila#le as
PC9 mastermiNes while others are a)aila#le as free dye "C 5ree% P"US a%d 9esolight ha)e
#ee% compared i% terms of #right%ess, cl$steri%g )aria#ility, a%d effect o% amplihcatio% C3
65rie)i%k a%d Stowell, 200.7 "C 5ree% P"US, SY20, a%d F)a5ree% ha)e #ee% compared
for their per1orma%ce i% heteroDygote detectio% 6=arrar et al, 20107 SY!9 5ree% * ca% #e $sed
for some applicatio%s where detectio% of heterod$pleNes is %ot %ecessary or desira#le
6Stepha%s et a. 200.7 2hese i%cl$de ge%otypi%g of mo%oploid orga%isms, methylatio%
a%alysis, a%d repeat typi%g ?owe)er, for all applicatio%s that de& pe%d o% heteroDygote
detectio%, a sat$rati%g 'NA dye with high heterod$pleN se%siti)ity is cr$cial
Instruments for CR?A
2he 1(rst of the i%str$me%ts capa#le of high&resol$tio% melti%g a%d with the appropriate software
algorithms was the ?9&1 6idaho 2ech%ology *%c7 2his i%str$me%t is desig%ed to work with
"igl(tCycler capillaries amplihed i% either the capillary "ightCycler *%str$me%ts 69oche Applied
Scie%ce7 or the 9apidCycler 2 6*daho 2ech%ology *%c7 #e1Mre #ei%g tra%sferred to the ?9&1
Samples are a%alysed o%e at a time so that testi%g large %$m#ers of samples is la#orio$s a%d more
pro%e to h$ma% error 68a%derstee% et al, 200-7 2his i%str$me%t was followed #y the "ightSca%%er
6idaho 2ech%ology *%c7C this time $si%g ,S& well or R./&well plates with PC9 also performed o% a
separate i%str$me%t 2he 1(rst of the *%str$me%ts to com#i%e real&time PC9 amplihcatio% a%d high&
resol$tio% melti%g a%alysis were the Cor#ett 6%ow ìage%7 9otorge%e S000 a%d the "ightCycler /.0
69oche Applied Scie%ce7 2he com#i%atio% of real&time PC9 a%d ?9<A pro)ides a $se1vl 3$ality
check of
Microtluidic Emulsion PCR
'. Reginald Beer and !o$n H. ;eamon
A#stract
PC9 has traQitio%ally #ee% performed i% microlitre&scale reactio%s #eca$se larger scale
)ol$mes are prohi#iti)ely eNpe%si)e a%d wastef$l while the smaller scales 6%a%olitre a%d
#elo))7 are impractical with a)aila#le sample ha%dli%g tools a%d detectio% Systems At the
microlitre scale, samples ca% co%tai% m$t$ally competiti)e a%d disti%ct targets, i%trod$ci%g
amplihcatio% #ias a%d competiti)e i%hi#itio% that degrade assay per1Mrma%ce <icro1l$iQic
Fm$lsio% PC9 has emerged as a tech%i3$e to resol)e these challe%ges #y a com#i%atio% of
two e%a#li%g tech%ologies Fm$lsio% PC9 pro)ides the ad)a%tages of fl$id partitio%i%g,
%amely elimi%atio% of sample #ias a%d the a#ility to r$% millio%s of reactio%s i% discrete
)ol$mes, while micro1l$idics sim$lta%eo$sly red$ces the sample )ol$me, i%trod$ces a le)el of
co%trol o)er em$lsio% parameterSC a%d pro)ides optical o#ser)a#ility of the partitio%ed
microreactors =$rthermore, si%ce micro1l$idic em$lsio%s ca% #e made mo%odisperse i% siDe,
they allow the ass$mptio% of a% a)erage dil$tio% per reactor to permit the eNploita& tio% of
Poisso% statistics for )ery acc$rate titre estimatio% <icro1l$idic em$lsio%s ca% also #e
employed to per1orm solid&phase amplificatio% with #ead&#ased assaySC com#|ii%g yet
a%other $se1$l tech%i3$e with the sample partitio%i%g #e%efits of droplets Ie eNpect the
ad)a%tages of #oth em$lsio% PC9 a%d micro1l$idics will e%co$rage %ew applicatio%s a%d the
i%tegratio% of these e%a#li%g tech%ologies will impro)e PC9 performa%ce
Emulsion PCR
2raditio%allyC PC9 has #ee% per1ormed i% microlitre&scale reactio%s #eca$se larger scale
6millilitre a%d a#o)e7 is prohi#iti)ely eNpe%si)e a%d wastef$l while the smaller scale 6%a%o&
litre a%d #elow7 is $%ser)ed #y a)aila#le sample ha%dli%g tools 'espite the lack of a)aila#le
tools, howe)er, the aQ)a%tages of goi%g to smaller siDes & %amely less reage%t $se a%d the
a#ility to approach si%gle copy a%alyses, #ecko%ed researchers to i%)estigate %o%&traditio%al
methods H%e of these methods has e%tered the mai%stream of %$cleic acids a%alysis, the $se
of em$lsio%s i% PC9
Fm$lsio%s are miNt$res of two or more immisci#le li3$ids A commo% eNample is oil a%d
))ater, #$t e)e% mayo%%aise is a% em$lsio% 6oil, )i%egar, egg yolks, etc7 Fm$lsio%s are two&
phase Systems of colloids where #oth phases are li3$id !eca$se they do %ot #le%Q, the
dispersed phase forms droplet&like compartme%ts withi% the co%ti%$o$s phase *t is this
compartme%taliDatio% that partitio%s a% a3$eo$s sample especially well for assays s$ch as
PC9 Prior to digital micro1l$idics a%d #atch&ge%erated em$lsio%s, the o%ly a)aila#le
^
of 00V 6o%e copy for e)ery two wells of sol$tio% o% a microarray plate, or e3$i)ale%tly o%e
copy for e)ery two droplets7, 7 V 00 U%der Sta%dard dil$tio% chemistry o%e might eNpect half
of the wells or droplets to ha)e positi)e reactio%s *% fact o%ly &R,RV will #e positi)e
accordi%g to Poisso% statistics 2his is #eca$se some wells get Dero copies, others will get o%e
copy, a small perce%tage will get two copies, a%d so o% 2his e1Kect is demo%strated i% =igs
11R a%d 11/ for 1 a%d 00 copies per droplet respecti)ely
2h$s we see a% importa%t #e%e1it of digital PC9+ we ca% i%fer starti%g copy co%ce%tra& tio%
#ased o% the perce%tage of droplets that Wgo hotX for the Poisso% regime a%d #ased o% Ct )al$es
for the greater tha% o%e copy per reactor dil$tio%s 6see =ig 1107 2his co%)e%ie%t statistical
o#ser)atio% compares fa)o$ra#ly to traditio%al 3PC9 methods which re3$ire the $ser to do a
time&co%s$mi%g cali#ratio% titratio% series o)er three or fo$r mag%it$des of starti%g copy
co%ce%tratio% with a well&k%ow% Sta%dard a%d meas$re the Ct shi1t for each Ie ha)e show%
pre)io$sly the acc$racy of digital PC9 i% a co%ti%$o$s flow micro1l$idic plat1orm, which is
displayed i% #elow Note the #l$e c$r)e is the predicted perce%tage of droplets that s$pport
ampli1(catio% )erses starti%g ge%omic template copies per droplet accordi%g the Poisso%
statistics 2racki%g the shape of this c$r)e are the eNperime%tal res$lts of 1S,000 droplets per
data poi%t at titratio%&3$a%ti1ied starti%g copy %$m#ers 2he displayed eNcelle%t agreeme%t
poi%ts to the power of this methoQ
Emulsitied solid&phase amplitication
Com#i%i%g em$lsi1icatio% with solid&phase amplihcatio% a)oids some #$lk amplihcatio%
limitatio%s descri#ed pre)io$sly, a%d pro)ides se)eral key ad)a%tages '$e to the large %$m#er
of droplets ge%erated #y em$lsi1katio%, template co%ce%tratio%s ca% #e dil$ted dow% to le)els
where the a)erage droplet co%tai%s, o% a)erage, less tha% a si%gle template
0 / 1 1 1 1 * *
N$mhtr 1 'NA ti<%}Ul~4S i% v%e drol%1
"igure ##$' &istri#ution of genomic co%ies in dro%lets under a Poisson %rocess for an a,erage
dilution of one co%y %er dro%let. 'ote t$at a%%ro-imately )AT of dro%lets are em%ty due to
some dro%lets recei,ing more t$an one co%y.
222 I Beer and
Microtluidic Emulsion PCR I %%2
i% sol$tio% witho$t a solid&phase compo%e%t, a%d to date "A2F&PC9has %ot #ee% $tiliDed for
solid&phase capt$re or emPC9
F%ha%ced solid&phase PC9 6FSP&PC976Kha% et al, 200.7 refers to PC9 primer opti&
miDatio% desig%ed speci1(cally for solid&phase ampli1icatio%s Kha% et al. 6200.7 tested a%
impro)ed primer desig% o% silica microspheres that i%creased ampli1(catio% e1hcie%cy a%d
yield #y employi%g %ested primers, there#y deco$pli%g the sol$tio% a%d solid phase primer
ki%etics 2he a%%eali%g temperat$res ))ere also co%trolled to e%s$re that limiti%g primer diQ%Xt
decrease i% e1hcie%cy with red$ced co%ce%tratio%, with sol$tio% phase primers possessi%g a
lower 2
m
tha% the solid phase :si%g these impro)eme%ts, Kha% et al 6200.7 were a#le to
impro)e soliQ&phase ampli1icatio% from 1/& to ,.&fold across three separate diag%ostic
targets
Carmo% et al 620027 i%)estigated %o%&em$lsified soliQ&phase PC9 i% microwells a%d fo$%Q
optimal ampli1(catio% whe% the primers were dista%ced from the #i%di%g moieties #y fi)e to
te% ?F5 6heNaethyle%e glycol7 spacers with a% approNimate le%gth of ,0&1.0 %$cleotides
U%der these co%ditio%s ro$ghly 20V ofthe immo#iliDed primers were eNte%ded6Carmo% et a.
'22'), sig%i1(ca%tly more tha% the 0RV eNte%sio% of oligo&dt6 107 primers co$pled to glass
slides o#tai%ed #y Adeesi et al. 6Adeesi et al, 2000C Kohsaka a%d Carso%, 1,,/7 Carmo% et al
fo$%d that the ma@ority of the ampli1katio% i%e1hcie%cies i% ampli1(catio% were d$e to steric
hi%dra%ce of the Taq polymerase #i%di%g to the immo#i& liDed template6Carmo% et al. 20027 as
opposed to steric hi%dra%ce of the #i%di%g of the template to the immo#iliDed primer as had
#ee% s$ggested #y others 65$o et al, 1,,/C Shchepi%o vetal, 1,,-7
Enric$ment
As disc$ssed pre)io$sly, the massi)e thro$ghp$t i%here%t i% em$lsihed reactio%s e%a#les the
$se of limiti%g dil$tio% to pro)ide si%gle molec$le amplihcatio% Iith millio%s to #il& lio%s of
reactio%s ge%erated per t$#e, 'NA co%ce%tratio%s ca% #e lo))ered to le)els ))here of the
droplets co%tai%i%g 'NA template, si%gle template droplets are predomi%a%t 2his allows
clo%al amplihcatio% witho$t competitio% #etwee% templates ?o))e)er, limit& i%g dil$tio%s
yield a large perce%tage of empty droplets with %o 'NA template #$t may co%tai% a 'NA
capt$re #ead %o%etheless Scree%i%g the 'NA capt$re #eads from these %o%&templated
droplets ))o$lQ #e eNtremely i%e1Kicie%t or eNpe%si)e for some applicatio%s, s$ch as ge%omic
se3$e%ci%g
Se)eral methods ha)e #ee% de)eloped to e%rich the #ead pop$latio% for those co)ered with amplico%
a%d discard the empty $%i%1ormati)e #eads !FA<i%g 6'ressma% et al, 200R7 $tiliDed hy#ridiDatio%
of 1l$oresce%t pro#es compleme%tary to k%ow% allelic )aria& tio%s o% the immo#iliDed templates to
select amplihed #eads )ia =ACS Strepta)idi%&coated paramag%etic #eads tethered to the templates
o% 'NA capt$re #eads )ia hy#ridiDed #ioti& %ylated were $sed #y <arg$iles et al 620007 to
selecti)ely retai% the amplico% coated 'NA capt$re #eads She%d$re et al. 620007 e1Kecti)ely re)ersed
this process #y #i%di%g large, %o%&mag%etic #eads to amplico%&coated positi)e #eads )ia #ioti%ylated
pro#es 2he ampli& co% #eads were the% selected #y ce%tri1$gatio% dow% a de%sity gradie%t, with $%&
templated #eads si%ki%g dow% the gradie%t, while 'NA #eads #o$%d to the larger %o%&mag%etic
#eads 1loated at the top 9egardless of the methodology e%richme%t ofimmo#iliDed em$l& sio% PC9
prod$cts permits the e1hcie%t segregatio% a%d $tiliDatio% of clo%ally amplihed templates for a )ariety
of applicatio%s
6'$al7 hy#riQiDatio% pro#es ,, 10, 11, 1R, .R, ./, ,0, 10,, 202, 20S
E
Fm$lsi1ied solid&phase ampli1(catio% 222 em$lsihed 'NAcapt$re #eads 22/ e%richme%t 22- impro)ed primer
desig% 220 PC9e1?cie%cy 220
Fm$lsio% PC9 21-&22, easeo1$se 21, em$lsio% thermosta#ility 21. micro1l$idic approaches to emPC9 220
picolitre&scale droplets o%&chip 21,
F%Dyme selectio% ,2
F3$ili#ri$m a%d ki%etic paradigms of PC9 -
"
=l$oresce%t detectio% chemistry -,, .2
=l$oresce%t i%dicators .
I
geNorm a%alysis 1/2,1/S
C
?igh&resol$tio% melti%ga%alysis 11, 1/,201&21R applicatio%s 20- assaydesig% 20, assay optimiDatio% 210
dyesfor**9<A 20R ?9<A chemistries 20S *%str$me%ts for ?9<A 20R so1t))are algorithms 20/
tro$#leshooti%g 212 $se1$l we#sites 21R
?ydrolysis pro#es ,,12,1R,.R, ./, .S, .., 10S&10-, 201
1
*daho 2ech%ology S,,1,,R,100,20R,20/
*%hi#itio% 2R&0-,-/, .-,,0,10,, 10/, 100 i%hi#itors from sample isolatio% R2 i%hi#itors i% a%imal material R1
i%hi#itors i% soil a%d pla%t material R0 i%hi#itory characteristic of a sample RR operator&i%trod$ced
)ariatio% RR sample e1lect R0
*%hi#itio% specihc to 9NA R0
hdelity of re)erse tra%scriptio% 6927 reactio%s R0
;n siico amplico% predictio% ./, .0
?
<ag%esi$m co%ce%tratio% /0, S., S,, -2, .0, .,, ,0,,1,10.,10,
<elti%ga%alysis 0, -, ,&1-, S.
<icro1l$idic PC9 12, 1S <icro 9NA a%alysis 1,1
eNpressio% profili%gof mi9NAs from si%gle mo$se FS cells 1,S
NCode
*<
$%i)ersal mi9NA392&PC9 1,2 92&3PC9 methods $sed to 3$a%ti1y mi{lNAs 1,2
2a3<a%] micro9NA assays 1,/ 2a3<a%] micro9NA arrays 1,0 <*`F checklist 102
-
Na%o'rop R-, R., ..
p
PC9 assay desig% .0 PC9 detectio% chemistry ,, 'NA polymerase ,,,100 i%terca4ati%g 1l$orophore dyes
10/ 92 primi%g strategies 102, 10R Poisso% statistics a%d small )ol$me 3PC9 221 Pre&PC9 steps a%d
optimiDatio% of 9NA 3$ality ,- P9FLCF"&` /.
Primer a%d pro#e modihcatio%s -,, .S Primer a%d pro#e 3$ality a%d co%ce%tratio% ,0, ,1, 10., 10,
Primer desig% programs ./,1S/ Primer optimiDatio% 112 Primer&pro#e Systems 100,10S
hydrolysis pro#es ,,1R, 10S,10- %o%&li%ear pro#es 10S oligo pro#es 10S,10-
i
3PC9a%dm$sic 02
R
9apid cycle PC9 0&-,1R&10 9apid temperat$re cycli%g 0,11 9eal&time PC9 i%str$me%tatio% 11, 12, 11,, 1RS
234 I
A!* --00, 11&1R
high&resol$tio% melti%gcapa#ilities 1R0 i%str$me%t siDe 1RS
"ightCycler -&1R,.2,,R, 10S, 111, 11/, 110, 120,121,1RR&1RS,20R m$ltipleN 1R0 optical Systems 1R0 price
1R- sample 1ormat 1R0 software 1R2 thermal System 120 thro$ghp$t 1R0
9eal&time 3PC9 a%alysis programs 1S/ 9elere%ce ge%e selectio% 1/1 9e)erse tra%scriptase R,20,20,R0, R.,
/0, 0S, -0, 101,10/,100,10S,1,/
234 I

PCR Troubleshooting and Optimization

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