Polymerised Chain Reaction

INTRODUCTION
  • PCR is a method of enzymatic amplification of a target sequence of DNA.
  • It is sensitive, selective (specific) and extremely rapid means of amplifying any desired sequence of double stranded DNA, which can be as short as 50-100 base pairs (bp) and as long as 10 kbp.
  • In PCR, the DNA to be amplified is replicated by DNA polymerase of Thermus aquaticus (Taq).
  • Taq polymerase is used because it is thermostable, not denatured at a temperature upto 95°C (in PCR DNA is to be heated to 94°-95° C for separation of strands).
Materials required:
  • Two primers, each about 20-35 bases long with sequence complementary to the sequence immediately adjacent to the DNA segment of interest ( flanking sequence) .
  • Divalent cations, magnesium or manganese ions; generally Mg2+ is used,
  • DNA polymerase (e.g., Taq polymerase) which can sustain high temperature (> 600 C).
  • A large number of free deoxynucleotides (dNTPs)
  • The target DNA fragment.
METHOD:
  • PCR uses DNA polymerase to repetitively amlify targeted portion of DNA.
  • Each cycle doubles the amout of DNA in the sample, leading to exponential increase with repeated cycles of amplification. 
  • SYBR Green Dye is used monitor PCR reactions 
  • Thus amplification after 'n' number of cycle in (2)". 
  • Twenty cycles provide an amplification of 106 (million) and 30 cycles of 109 (billion).
  • PCR occurs in following steps -A second cycle of cooling, allows further binding of the primers and a second round of polymerization is repeated. The process of heating (denaturation), cooling (annealing) and polymerization is repeated many times.
  1. Isolation of target DNA sequence :- A DNA containing the sequences to be amplified is isolated.
  2. Primers construction:- The sequence of flanking region (nucleotide sequence of short segments on each side of target DNA sequence) are used to construct 2 single stranded oligonucleotides which are complementary to the respective flanking region. These oligonucleotides functions as primers in PCR reaction.
  3. Denaturation of DNA :- Target DNA is heated to separate the double strand of DNA into single strands.
  4. Annealing of primers to single stranded DNA :- The separated strands are cooled and allowed to anneal to the two primers, one for each strand.
  5. Chain extension :- DNA polymerase and deoxyribonucleotides (dNTP) are added to the mixture. DNA polymerase (Taq polymerase) then uses the primer for DNA synthesis using target strands as templates. On both strands, Primer is amplified in 5'-43' direction by addition of nucleotides (dNTP) at 3' end. The mixture is once again heated to melt (separate) all double stranded structure. There are now four strands of DNA.
  • As Taq polymerase is not denatured on heating and therefore does not have to be added at each successive cycle.
  • Thus following are required in PCR :- Target double stranded DNA, two specific primers, a thermostable DNA polymerase (Taq polymerase), deoxyribonucleotides (dNTP).
USES:
  • The most sensitive method for detecting cervical chlamydia trachomatis infection
  • Prenatal diagnosis of Hemophilia 
  • HIV can be detected and confirmed
Advantages:
  • The major advantage of PCR over cloning as a mechanism for amplifying a specific DNA sequence are
  • sensitivity : DNA sequences present in only trace amounts can be amplified to become the predominant sequence
  • speed
  • less technically difficult than traditional cloning methods using recombinant DNA techniques. Disadvantages:
  • To synthesize primers, we need to know the sequence flanking the DNA segment of interest.
  • Applies only to short DNA fragments, typically less than 10 kb.
Exam Question
  • PCR is the most sensitive method for detecting cervical chlamydia trachomatis infection
  • Cation used in PCR is Magnesium
  • Primer, Taq polmerase, & dNTPS are the material required for PCR
  • SYBR Green Dye is used for PCR
  • PCR is used for Prenatal diagnosis of Hemophilia 
  • PCR is an exponential , specific technique Carried out by thermostable DNA polymerase
  • In PCR Annealing is done after DNA denaturation
  • In PCR Acquaticus thermophilus is preferred over E.coli. because Thermostable at temperature at which DNA liquefies
  • HIV can be detected and confirmed by PCR

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