In this article we will discuss about:- 1. Introduction to PCR 2. Technique of PCR 3. Modified Forms.
Introduction to PCR:
PCR provides a simple and ingenious method for exponential amplification of specific DNA sequences by in vitro DNA synthesis, i.e., this technique has made it possible to synthesize large quantities of DNA fragments without cloning it. Kary Mulis in 1985 developed the technique based on the use of an enzyme which is named as Taq DNA polymerase.
The PCR technique has now been automated and is carried out by a specially designed machine.
Technique of PCR:
The technique involves the following three steps (Fig. 18.16):
I. Denaturation of DNA Fragment:
The target DNA containing sequence to be amplified is heat-denatured (around 94°C for 15 sec) to separate its complementary strands, this process is called melting of target DNA.
II. Annealing of Primers:
Primers are added in excess and the temperature is lowered to about 68°C for 60 sec., as a result the primers form the hydrogen bonds and anneal to the DNA on both sides of the DNA sequence.
III. Primer Extension:
Finally different nucleoside triphosphate (dATP, dCTP, dCTP, dTTP) and a thermo-stable DNA polymerase (Taq polymerase from Thermus aquaticus and Vent polymerase from Thermococcus litoralis) are added to the reaction mixture, it helps in polymerization process of primers and, therefore, extends the primers (at 68°C) resulting in synthesis of multiple copies of target DNA sequence.
After completion of all these steps in one cycle, again the second cycle is repeated following the same process. If 20 such cycles occur, then about one million copies of target DNA sequence are produced. Recently this technology has been improved much more, where instead of Taq polymerase the rTth polymerase is used which transcribe RNA to DNA, and thereafter amplify the DNA.
Modified Forms of PCR:
The conventional PCR is the symmetrical PCR technique.
There are some other modified forms of PCR which used for various purposes:
AP-PCR (Arbitrarily Primed Polymerase Chain Reaction):
It requires only a single primer of relatively much smaller length compared to the primers used in PCR. This technique is used for DNA profiling, in animal and plant biotechnology as well as in forensic medicine.
Target sequences of one strand may be amplified in several order of magnitude more as compared to its complementary strand. This approach is particularly useful for generating single stranded DNA fragment to be used for sequencing of DNA.
IPCR (Inverted Polymerase Chain Reaction):
In this method it allows the amplification of DNA, flanking a known DNA sequence, the primers are facing outwards. Using the inverse PCR, the unknown sequences flanking known sequences can be readily amplified.
RT-PCR (Reverse Transcriptase Polymerase Chain Reaction):
Although the PCR amplification is generally performed on the DNA template but using this technique the RNA also can be used for amplification. This technique is particularly useful for studying the expression of genes and for monitoring the obscure species of mRNA.
Nested PCR primers are ones that are internal to the first primer pair. The larger fragments produced by the first round of PCR is used as the template for the second PCR. This technique eliminates any spurious nonspecific amplification products.
Application of PCR in Biotechnology: PCR has many fold applications.
i. The amplification of gene fragments as fast alternative of cloning:
(a) Inserts of bacterial plasmids can be amplified with primers.
(b) DNA from known sequence can be obtained by designing primers.
(c) PCR helps in identification of homologous sequences from related organisms.
(d) Using RT-PCR the 3′ end of cDNA can be amplified (RACE: Rapid Amplification of cDNA Ends).
(e) Reverse PCR helps to know the flanking sequences of a known DNA clone.
ii. Modification of DNA fragments:
Site directed mutagenesis using oligonucleotides as PCR primers provides a powerful approach to study structure-function relation.
iii. Diagnosis of pathogenic microorganism:
DNA from the infected parts of a person or animal may be subjected to PCR with primer specific gene of the pathogen and diagnosis can be done on amplification of DNA.
iv. DNA analysis of archaeological specimens:
As DNA is relatively stable and remain intact for a long period of time, PCR can help in analysis of DNA from those embedded materials.
v. Detection of mutation relevant for inherited diseases:
Any point mutation, a deletion or an insertion and expanded tandem trinucleotide repeat can be detected by PCR. Somatic mutations in oncogenes or tumour repressor genes can also be detected by PCR with primers flanking the insertions or deletions.
vi. Analysis of genetic markers for forensic applications, for paternity testing and for the mapping of hereditary traits.
(a) Amplification of SSR.
(b) RAPD (Random Amplified Polymorphic DNA) with arbitrary, often short (10 bp) primers.
vii. Species-specific amplification of DNA segments between interspersed repeat elements (IRS) using the primer based on the SINE sequence (Short Interspersed Nuclear Elements).
viii. Genetic Engineering using PCR:
Using PCR we can incorporate alteration or mutation in the ultimate product by choice altering, removing or adding sequences to the primer at the 5′ end.
By recombinant PCR technique, it is possible to join two DNA fragments at a specific site through complementary overlaps (This technique is termed as splicing). By synthesizing two mutagenic primers, spanning the internal site to be changed, it is possible to introduce mutations within a fragment.