Recombinant DNA Technology

1. Polymerase Chain
Reaction- Basic
Principle
Promila Sheoran
Ph.D. Biotechnology
GJU S&T Hisar

2. Principle of the PCR
The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of
copies of a gene.
The cycling reactions :
•There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is
done on an automated cycler, which can heat and cool the tubes with the reaction
mixture in a very short time.
1. Denaturation at 94°C :
During the denaturation, the double strand melts open to single stranded DNA,
all enzymatic reactions stop (for example : the extension from a previous cycle).
2. Annealing at 54°C :
The primers are jiggling around, caused by the Brownian motion. Ionic bonds are
constantly formed and broken between the single stranded primer and the single
stranded template. The more stable bonds last a little bit longer (primers that fit
exactly) and on that little piece of double stranded DNA (template and primer), the
polymerase can attach and starts copying the template. Once there are a few bases
built in, the ionic bond is so strong between the template and the primer, that it does
not break anymore.

3. 3. Extension at 72°C :
This is the ideal working temperature for the polymerase. The primers, where there
are a few bases built in, already have a stronger ionic attraction to the template than
the forces breaking these attractions. Primers that are on positions with no exact
match, get loose again (because of the higher temperature) and don't give an
extension of the fragment.
The bases (complementary to the template) are coupled to the primer on the 3' side
(the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side,
bases are added complementary to the template)

5. Inverse PCR
•Inverse PCR (IPCR) was designed for amplifying anonymous flanking genomic DNA
regions.
• The technique involves the digestion of source DNA, circulation of restriction
fragments, and amplification using oligonucleotides that prime the DNA synthesis
directed away from the core region of a known sequence, i.e., opposite of the direction
of primers used in normal or standard PCR.

6. •Prior to the invention of the polymerase chain reaction (PCR), the acquisition of a
specific DNA fragment usually entailed the construction and screening of DNA libraries,
and the traditional “walking” into flanking DNA fragments involved the successive
probing of libraries with clones obtained in the prior screening.
•These time-consuming procedures could be replaced by IPCR. Because IPCR can be used
to efficiently and rapidly amplify regions of unknown sequence flanking any identified
segment of cDNA or genomic DNA, researchers do not need to construct and screen DNA
libraries to obtain additional unidentified DNA sequence information using this
technique.
• Some recombinant phage or plasmids may be unstable in bacteria and amplified
libraries tend to lose them. IPCR eliminates this problem.

7. Anchored PCR
•When sequence of only one end of
the desired segment or gene is
known, the primer complementary
to the 3’ end of this strand is used
to produce several copies of only
one strand of the desired segment.
•Now a poly–G is added to the 3’end
(the end for which primer is not
available) of the single-strand DNA
copies produced by PCR.
•This allows the use of
complementary homopolymer, poly-
C, to be used as primer for copying
the DNA single-strands generated by
PCR, and give rise to the complete
DNA duplex that can be amplified
normally.
•This approach has been termed as
anchored PCR and the poly-G tail
added to the 3’ ends of the single
strand copies is called anchor.

8. Asymmetric PCR
•Asymmetric PCR is used to preferentially amplify one strand of the original DNA
more than the other.
•It finds use in some types of sequencing and hybridization probing where having
only one of the two complementary stands is ideal.
• PCR is carried out as usual, but with a great excess of the primers for the chosen
strand.
•Due to the slow (arithmetic) amplification later in the reaction after the limiting
primer has been used up, extra cycles of PCR are required

9. •To achieve this, quantities of the two
primers for the 3’ borders of the target
DNA segment are so adjusted in the
reaction mixture that one of them is
exhausted about 10 cycles or so cycles
before the PCR is terminated.
•As a result, in the terminal 10 or so
cycles only a single strand of DNA
segment is copied; these single-strand
copies are the starting materials for
DNA sequencing.
•This variation of PCR is called
asymmetric PCR.

10. RT-PCR
•This variation can be used to amplify RNA sequences into DNA duplexes. At first, a
cDNA copy of the RNA is produced using the enzyme reverse transcriptase; this
cDNA is then used for amplification.
•In case of eukaryotic mRNAs, an oligo-T sequence can serve as one of the primers
for the PCR as well as for the reverse transcription step.

11. •The mRNA is copied into a single
cDNA strand using an oligo-T as
primer, which pairs with the 3’ poly- A
tail of the mRNA.
•An oligo –G tail is now added to the
3’-end of the cDNA single-strand (the
sequence of this end of cDNA is not
known) by the enzyme terminal
deoxynucleotidyl transferase.
•The cDNA single-strand now has an
oligo-T sequence at one end (5’ end)
and an oligo-G sequence at the other
(3’ end).
•We now use an oligo-C as a primer to
copy the single-strand cDNA into cDNA
duplex, which is amplified using an
oligo-T and an oligo-C as primers.

12. PCR for site directed mutagenesis
•This technique is used for introducing mutations at the desired place in a DNA
sequence by altering the sequences of primers.
•Since mutations are introduced only through primers, mutations are limited to the
ends of the gene sequence.
•A variation of this technique allows mutations to any place of interest in the gene-
the method is described as overlap extension PCR.

13. (i) In two separate PCR reactions, a particular gene
is amplified into two separate segments. In both
reactions there is one primer at the end of the
gene and the other internal to the sequence.
(ii) The internal primers in two reactions are
complementary to one another, so that the
amplified products will have their ends internal
to the original sequence.
(iii) These internal ends of products in two reactions
will overlap. The sequences of internal primers
can be suitably modified to introduce alterations
in the overlap region.
(iv) The two PCR products are denatured and
annealed, so that the internal ends in the overlap
region will work as primers for each other.
Extension of these primers results in the
formation of a complex gene, with the mutation
incorporated into it

14. Thank You