Polymerase Chain Reaction (PCR)

Introduction

  • PCR: procedure that amplifies a specific sequence of DNA
  • In 1983, Kary Mullis at Cetus Corporation developed the polymerase chain reaction (PCR).
  • The objective of PCR is to produce a large amount of DNA in a test tube (in vitro), starting from only a trace amount.
  • In theory, only a single template strand is needed to copy and generate millions of new identical DNA molecules.
  • PCR has made an impact of four main areas of genetic research: gene mapping, cloning, DNA sequencing, and gene detection.
  • The development of PCR transformed molecular biology from a difficult science to one of the most accessible and widely used disciplines of biotechnology.

PCR Step by Step

  • PCR involves a repetitive series of cycles, each of which consists of template denaturation, primer annealing, and extension of the annealed primer by Taq DNA polymerase.
  • The template DNA, oligonucleotide primers, thermo- stable DNA polymerase (Taq), the four deoxynucleotides (A, T, G, C), and reaction buffer are mixed in a single micro centrifuge tube.
  • The tube is then placed into a thermal cycler.
  • Thermal cyclers: contain an aluminum block that holds the samples and can be rapidly heated and cooled across extreme temperature differences.
  • Temperature/thermal cycling: the rapid heating and cooling of this thermal block.
  • Denaturation step: the thermal cycler heats the sample to 94°C, so the template strands separate (denature).
  • Annealing step: the thermal cycler then rapidly cools to 60°C to allow the primers to anneal to the separated template strands; the two original template strands may reanneal to each other or compete with the primers for the primers complementary binding sites.
  • Extension step: the thermal cycler heats the sample to 72°C for Taq DNA polymerase to extend the primers and make complete copies of each template DNA strand.
  • Two new copies of each complementary strand are created. There are now two sets of double-stranded DNA (dsDNA). These two sets of dsDNA can now be used for another cycle and subsequent strand synthesis.
  • At this stage, a complete temperature cycle (thermal cycle) has been completed.
    • Temperature cycle = denaturation step + annealing step + extension step
  • Usually, thermal cycling continues for about 40 cycles.
  • After each thermal cycle, the number of template strands doubles, resulting in an exponential increase in the number of template DNA strands.
  • On the first cycle, the two primers anneal to the original genomic template DNA strands at opposite ends and on opposite strands.
  • After the first complete temperature cycle, two new strands are generated that are shorter than the original template strands, but still longer than the length of the DNA that the researcher wants to amplify.
  • It isn’t until the third thermal cycle that fragments of the precise length are generated.
  • It is the template strands of the precise length that are amplified exponentially (X^n, where X=the number of original template strands and n=the number of cycles).
  • There is always one set of original long-template DNA molecules, which is never fully duplicated.
  • After each thermal cycle, two intermediate-length strands are produced, but because they can only be generated from the original template strands, the intermediate strands are not exponentially amplified.
  • It is the precise-length strands generated from the intermediate strands that amplify exponentially at each cycle.