Berbagai Teknik Dalam Rekayasa Genetika: Drs. Sutarno, MSC., PHD

Berbagai teknik dalam
Rekayasa Genetika
Drs. Sutarno, MSc., PhD.
 Rekayasa genetika merupakan disiplin “baru” yang
keberadaannya dimulai dari perkembangan teknik-
teknik molekuler sejak tahun 1960an, yaitu sejak
ditemukannya susunan kimia asam
deoksiribonukleat (double helix) pada inti sel.
 Sejak saat itu, pusat-pusat penelitian baik milik
pemerintah maupun swasta berlomba-lomba
melakukan pemetaan gen dari sejumlah tanaman
dan hewan dengan tujuan untuk memindahkan sifat-
sifatnya ke tanaman atau hewan lain.
 Berbagai teknik biologi molekuler kemudian
berkembang secara pesat, diantaranya teknik
ekstraksi DNA, elektroforesis, hibridisasi asam
nukleat, sekuensing DNA, Polymerase Chain
Reaction (PCR) dan lain sebagainya.
 Sejakditemukan PCR, berkembang
berbagai teknik berbasis PCR (PCR based
techniques), seperti:
 Polymerase Chain Reaction-Restriction
Fragment Length Polymorphism (PCR-RFLP),
 Random amplified polymorphic DNA (RAPD),
 Amplified fragment length polymorphisms

(AFLPs),
 Single nucleotide polymorphisms (SNPs),
 Microsatellites dll.
 Meskipun tujuan akhir dr rekayasa genetika
adalah umumnya berupa ekspresi gen pada
inang, kenyataannya, sebagian besar teknik dan
waktu dalam rekayasa genetika digunakan
untuk mengisolasi gen dan kemudian
meng’klon’kannya.
Berbagai teknik yang digunakan dalam
rekayasa genetika
1. Complementary DNA
 Complementary DNA (cDNA) is DNA made from

mRNA.
 This makes use of the enzyme reverse
transcriptase, which does the reverse of
transcription: it synthesises DNA from an RNA
template.
 It is produced naturally by a group of viruses
called the retroviruses (which include HIV), and
it helps them to invade cells.
 In genetic engineering reverse transcriptase is
used to make an artificial gene of cDNA.
making an artificial gene of
cDNA.
cDNA has helped to solve different
problems in genetic engineering:
 It makes genes much easier to find. There are
some 70 000 genes in the human genome, and
finding one gene out of this many is a very
difficult (though not impossible) task.
 However a given cell only expresses a few
genes, so only makes a few different kinds of
mRNA molecule.
 For example the b cells of the pancreas make
insulin, so make lots of mRNA molecules coding
for insulin. This mRNA can be isolated from
these cells and used to make cDNA of the insulin
gene.
2. Restriction Enzymes
 These are enzymes that cut DNA at specific sites.
 They are properly called restriction endonucleases
because they cut the bonds in the middle of the
polynucleotide chain.
 Their biochemical activity is the hydrolysis ("digestion")
of the phosphodiester backbone at specific sites in a
DNA sequence.
 Some restriction enzymes cut straight across both
chains, forming blunt ends, but most enzymes make a
staggered cut in the two strands, forming sticky ends.
 The cut ends are “sticky” because they have short
stretches of single-stranded DNA with complementary
sequences. These sticky ends will stick (or anneal) to
another piece of DNA by complementary base pairing,
but only if they have both been cut with the same
restriction enzyme.
 Enzim restriksi biasanya diisolasi dari bakteri
 Yang digunakan dalam rekayasa genetika: enzim
restriksi endonuklease, enzim ini mengenali DNA pada
situs khusus dan memotong pada situs tsb.
 Situs pengenalan ER adalah daerah yg simetri atau
disebut dg palindrom, artinya bila kedua utas DNA tsb
masing-masing dibaca dg arah yg sama akan
memberikan urutan nukleotida yg sama.
 Pemotongan ER menghasilkan dua jenis ujung
potongan: berujung rata/ blunt end, dan berujung tidak
rata/sticky end/ cohesive. ER yg memotong pada pusat
palindrom akan menghasilkan potongan berujung rata,
sedangkan ER yang memotong diluar pusat simetri
menghasilkan potongan berujung kohesif
 Restriction enzymes are highly specific, and will only cut DNA
at specific base sequences, 4-8 base pairs long, called
recognition sequences.
 Restriction enzymes are produced naturally by bacteria as a
defence against viruses (they “restrict” viral growth), but they
are enormously useful in genetic engineering for cutting DNA
at precise places ("molecular scissors"). Short lengths of
DNA cut out by restriction enzymes are called restriction
fragments. There are thousands of different restriction
enzymes known, with over a hundred different recognition
sequences.
 Restriction enzymes are named after the bacteria species
they came from, so EcoR1 is from E. coli strain R, HindIII is
from Haemophilis influenzae.
3. DNA Ligase
 Enzim ligase menyambung dua ujung DNA melalui ikatan
kovalen antara ujung 3’OH dari utas yang satu dengan ujung
5’P dari utas yang lain.
 2 tipe enzim ligase yg sering digunakan: DNA ligase dari
E.coli, dan DNA ligase dari fage T4.
 Ujung kohesif hanya dpt disambung dg ujung kohesif yg
kompatibel dan memenuhi komplementaritas (A-T dan G-C).
Ujung kohesif lebih efisien dlm penyambungan.
 This enzyme repairs broken DNA by joining two nucleotides
in a DNA strand. It is commonly used in genetic engineering
to do the reverse of a restriction enzyme, i.e. to join together
complementary restriction fragments.
3. DNA Ligase
 The sticky ends allow two complementary restriction
fragments to anneal, but only by weak hydrogen
bonds, which can quite easily be broken, say by
gentle heating. The backbone is still incomplete.
 DNA ligase completes the DNA backbone by forming
covalent bonds. Restriction enzymes and DNA ligase
can therefore be used together to join lengths of DNA
from different sources.
Kerja Enzim DNA Ligase

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Berbagai teknik dalam

 Amplified fragment length polymorphisms

 Complementary DNA (cDNA) is DNA made from

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