Identification and selection of

the right clones

 DNA Cloning:
 It is a technique for reproducing
DNA fragments. 
 Achieved by two different
approaches: 
▪ Cell Based
  ▪ Using PCR. 
 Amplifying a specific piece of DNA
via a bacterial cell
 Vector is required to carry the
DNA fragment of interest into
the host cell. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin
Cummings
Cloning Vector:
 Five Steps in Cloning DNA
Isolation of genomic DNA and vector

Cleavage of DNA and vector by RE’s.

Ligation

Transformation of rDNA in to bacteria

Ref: http://cnx.org

Identify the right clone.

 Gene libraries
 It is a collection of recombinant clones representing the
entire genome of an organism.

 Each clone is like a book in that library containing part of

the information contained in the genome.

 It is a collection of cloned DNA fragments. 

 Types of Gene libraries:
• Two types-depending upon the source of DNA used

Genomic library cDNA library

Contains DNA fragments Contains cDNA molecules

representing the entire synthesized from mRNA
genome of an organism molecules in a cell
 Screening DNA Libraries

 The process of determining which of those clones carry the gene

of interest in.
 Possible bacterial clone products:
 Non transformed
 Transformed one without the gene of interest.
 Transformed one with gene of interest
 Methods for screening of Transformed Cells

Selectable Marker Based

Use of Chromogenic Substrates

Hybridization Based Screening

Immunological Screening

PCR Based Screening

In vitro Translation Based Screening

Screening by complementation
1. Selectable Marker Genes
 Selectable markers :

• Used to recover transformed cells from a sea of non-

transformed cells

• Allows the selection of transformed cells - by their ability

to grow in the presence of selection pressure
 1.1. Antibiotic Resistance Markers

a) Neomycin Phosphotransferase II (npt

II)

b) Hygromycin Phosphotransferase (hpt)

c) Gentamycin Acetyltransferase

d) Streptomycin & Spectinomycin

 A. Neomycin Phosphotransferase II
Source :
• npt II = neomycin phosphotransferase II Tn5
Transposon
It inactivates a number of aminoglycoside antibiotics
eg: Kanamycin, Neomycin, Geneticin(G418), Paromycin
- They inhibits protein synthesis & protein translocation across membranes.
( Conc: 50 to 500mg/l)
• Expression of npt II gene results in synthesis of NPTII enzyme
•
Km NPTII enzyme Km-PO4
(Disrupts plant growth) (Unable to disrupts plant growth)
 B. Hygromycin Phosphotransferase Gene (hpt)

 Denoted hpt, hph or aphIV- derived from E.coli

 Hygromycin phosphotransferase (HPT) detoxifies the
aminoglycoside antibiotic hygromycin B 
 Hygromycin B(20-200mg/ml) is more sensitive than
Kanamycin
 Kills sensitive cells by interferes with protein synthesis
 C. Gentamycin Acetyltransferase Gene

• Gentamicin acetyl transferases detoxify aminoglycoside antibiotics

containing a amino group.

Acetyl-CoA + Gentamicin C CoA + N'-acetylgentamicin Enzyme:

Amino glycoside -3 - N- acetyltransferases (ACC)

• Detoxify Gentamycin, Neomycin, Kanamycin etc.

Antibiotics for negative selection:
Antibiotics for negative selection:

Source: www.promega.com • techserv@promega.com

 Insertional inactivation:
 More efficient method than the direct
selection.
 one of the genetic trait is disrupted by
inserting foreign DNA.
 pBR322 (ampr gene + tetr gene).
 If the target DNA is inserted into tetr gene
using Bam HI, the property of resistance to
tetracycline will be lost.
 Such recombinants would be test sensitive.
Screening bacteria by replica plating
2. Visual Screening
 Alpha-complementation
Click to edit Master title style

• Lac Z gene product β-galactosidase (1029 aa), gives rise to the

functional enzyme after tetramerization
• Tetramerization is dependent on the presence of the N-terminal region
spanning the first 50 residues(alpha peptide)
• Deletions in the N-terminal sequence generate a so-called omega
peptide (M15 E. Coli strain )
• N-terminal portion is added in trans restores the activity of the omega
peptide can be fully restored (Gallagher et al., 1994).

23
 Chromogenic Substrate of β-galactosidase :
 X-gal (5-bromo-4-chloro- 3-indolyl-D-galacto pyranoside)
 Inducer: IPTG (iso-propyl-thiogalactoside)

Spontaneous
Hydrolysis dimerization &
oxidation

5-bromo-4-chloro-3- Dibromo -dichloro- indigo, an

X-gal
Galactose hydroxyindole intensely blue product which is
(colourless)
insoluble
Blue white selection:
Blue white selection:
GFP_Reporter gene

• screening and identification of recombinant

clones is by using the green fluorescent protein
(GFP) from jellyfish Aequorea victoria.
• It is a reporter molecule for monitoring gene
expression, protein localization, protein-
protein interaction etc.
 pGREENscript A

• pGREENscript A in E. coli produced

colonies showing yellow color in
day light and strong green
fluorescence under long-UV.
• Inserted foreign genes are selected
on the basis of loss of the
fluorescence caused by inactivation
of the GFP production.
 Plaque phenotype

• Insertional inactivation of cI gene

• During an infection cycle, virus
undergoes a lytic and lysogenic stages.
• The cI gene encodes for cI repressor
which is responsible for the formation
of lysogens.
• In the presence of functional cI, the
plaques contains unlysed host cells and
has a turbid appearance where as in the
absence it will clear.
• This feature can be use to screen the clone to detect functional cI (absence
of clone) or absence of cI (presence of insert).
 3. Hybridization Based Screening
Methods
A. Colony Hybridization
B. Plaque-lift Hybridization:
 a. Colony Hybridization
 Nucleic acid hybridization
is the most commonly
used rapid method of
library screening.
 Developed by Grunstein
and Hogness (1975) .
 a. Colony Hybridization
Colonies replica plated on a nitrocellulose filter -
master plate is preserved

Lyse bacteria and denature DNA with denaturing

solution (NaOH and SDS),treat with Proteinase
K

Neutralize pH (NaCl and buffer pH7.4)

Fix ssDNA using UV light or baking at 80°C

Hybridize with labelled probe -Autoradiography

 b. Plaque-lift Hybridization:

• Benton and Davis (1977)

devised a method called
plaque lift.
• applied to the isolation of
recombinant phage by nucleic
acid hybridization
• The most widely applied
method of library screening.
4. Immunological screening
 4. Immunological screening
 Use Ab to detect desired clone
 Ab specifically recognize antigenic
determinants (epitope) on the
polypeptide synthesized by a target
clone.
 often used for screening cDNA
expression libraries

Source : Principles of Gene Manipulation :sixth edition By Sandy B. Primrose, Richard Twyman and Robert W Old
Immunological Screening of
Expressed genes:

Source: An Introduction to Genetic Engineering By Desmond S. T. Nicholl

 Screening of cDNA libraries in expression vector
λgt11.

• The cDNA inserted upstream from the

gene for β-galactosidase will give rise to
a fusion protein under induction of
IPTG.
• The plaques are then blotted onto a
nylon membrane filter and probed with
an antibody specific for the protein
coded for by the cDNA.
• A secondary labelled antibody directed
to the specific antibody can then be
used to identify the location (plaque) of
the cDNA.

Source: Principles and Techniques of Biochemistry and Molecular Biology By Keith Wilson, John Walker
5.PCR Based Screening :
 PCR screening of gene libraries
 library screening by PCR is
demonstrated by Takumi and Lodish
(1994)
 It involves lysing the bacteria and
amplifying a portion of the plasmid.
 You can use either insert specific
primers or vector-specific primers to
screen for recombinant plasmids.
 Advantage :Rapidity of the technique :
screening may be undertaken in 3–4 h.
PCR screening of gene libraries

Source: Principles and Techniques of Biochemistry and Molecular Biology

By Keith Wilson, John Walker
 Colony PCR
 Primers may be insert-specific, vector-specific, or
both to detect the insert.
 To determine the orientation of the insert, a
combination of vector-specific and insert-specific
primers is recommended.
 Colony screening by PCR is suitable for inserts
shorter than 3 kb.
Designing Colony PCR Primers
Analyzing PCR Product Size on a
Gel
 6. Detection based on in vitro
translation of mRNA

a)  Hybrid released translation (HRT)
b) Hybrid arrested translation (HART)
 Hybrid Release Translation (HRT) and
Hybrid Arrest Translation (HART)
• HRT and HART can identify the
Translation Product of a Cloned Gene
• In vitro translation system (wheat
germ or rabbit reticulocytes).
• Use a radioactive amino acid
([35S]methionine)
• SDS-PAGE
• Autoradiography
 Hybrid released translation (HRT)
 mRNAs homologous to cDNA clones
are specifically trapped as RNA/DNA
hybrids on nitrocelloulose filters.
 Remove nonspecifically bound RNA:
By washing at specific temperatures
and salt concentrations
 Release hybridized mRNA by boiling
the filter, precipitated with ethanol,
and translated in vitro. 
  Hybrid released translation (HRT) & Hybrid arrested
translation (HART).

HRT/HST HART
 mRNAs homologous to cDNA  Total mRNA are incubated with
clones are specifically trapped as melted DNA constructs from
RNA/DNA hybrids on recombinant cells.
nitrocelloulose filters  Hybridization prevents the
 Remove the mRNA translation of specific mRNA in a
cell-free system
 In vitro translation of the selected
mRNA  Identifies a gene product by its
disappearance from the pattern of
 Translation reveals one or a few translation products.
polypeptides
 Hybrid Hybrid arrested translation
released translation (HRT) (HART)
 9. Other methods for Analysis
of cloned genes
 Nucleic acid hybridization
 Southern blotting
 Colony hybridization
 Dot blot.
 Nucleic acid hybridization
 Hybridization- utilizes "probe
sequence" , and when it finds
its intended target, binds to it
by Watson-Crick base pairing.
 Since the "probe" is attached
to a label such as a fluorescent
chemical , the bound sequence
can thus be visualized.
 Blotting
 Blotting describes the
immobilization of sample
nucleic acids on to a solid
support, generally nylon or
nitrocellulose membranes.
 The blotted nucleic acids are
then used as ‘targets’ in
subsequent hybridization
experiments
 Southern blotting

 1975 Edward Southern developed this

technique.
 detecting fragments in an agarose gel that are
complementary to a given RNA or DNA
sequence.
 Southern blot
 Southern blot analysis reveals information about DNA
identity, size, and abundance.
 It involves
 separating DNA fragments based on size via
electrophoresis,
 transferring them to a membrane((up to 15 kb in length
taking around 18hours or overnight.)
 hybridization with a labelled sequence-specific probe,
 washing, and
 finally detection of labelled DNA band(s). 
  Dot Blot and Slot Blot
Hybridization
 A technique for detecting, analyzing, and identifying
proteins, similar to the western blot technique but
differing in that protein samples are not separated
electrophoretically but are spotted through circular
templates directly onto the membrane or paper
substrate.
 Dot Blot and Slot Blot
 The simplification of Southern and Western blots
saving the time involved in procedures of
chromatography, electrophoresis, restriction
digestion and blotting of DNA or proteins from the
gel to membrane.
 nucleic acid mixture is directly applied (blotted) on to the nylon or
nitrocellulose membrane where hybridization between probe and target
takes place, denatured to single-stranded form and baked at 80°C to bind
DNA target to membrane.
 Dot-blot, target is blotted as circular blots whereas in slot-blots, it is in the
form of rectangular blots.
 slot-blot offers greater precision in observing different hybridization signals.
 After blotting, membrane is allowed to dry and non-specific sites are
blocked by soaking in blocking buffer containing BSA.
 It is then followed by hybridization of labeled probe for detection of specific
sequences or gene.
 Summary:

Source : Principles of Gene Manipulation :sixth edition By Sandy B. Primrose, Richard Twyman and Robert W Old
 Reference:
 Recombinant DNA & Biotechnology by Kreuzer, H., Massey,
A.,2001,, ASM Press, Washington
 Principles of Gene Manipulation :sixth edition By Sandy B.
Primrose, Richard Twyman and Robert W Old
 Principles and Techniques of Biochemistry and Molecular
Biology By Keith Wilson, John Walker
 Gene Cloning and DNA Analysis: An Introduction, Sixth
Edition. 2010. Brown TA. Blackwell Science Ltd., Oxford.