Fundamentals of the Polymerase Chain Reaction

Of all the technical advances in modern molecular biology, the polymerase chain reaction (PCR) is one of the most useful. The PCR provides a means of amplifying DNA sequences. Starting with incredibly tiny amounts of any particular DNA molecule, the PCR can be used to generate microgram quantities of DNA. PCR is sufficiently sensitive that it can amplify the DNA from a single cell into amounts sufficient for cloning or sequencing. Consequently, PCR is used in clinical diagnosis, genetic analysis, genetic engineering and forensic analysis. In particular, PCR has revolutionized and speeded up the whole area of recombinant DNA technology. Previously, cloned DNA was made by growing up bacterial cultures and extracting and purifying the DNA. PCR allows the rapid generation of large amounts of specific DNA sequences that are easier to purify and less damaged. In this chapter we will examine how the polymerase chain reaction works. As the name indicates, DNA polymerase is used to manufacture DNA using a pre-existing DNA molecule as template. Each new DNA molecule synthesized becomes a template for generating more, thus creating a chain reaction. The PCR actually amplifies only a chosen segment (the target sequence) within the original DNA template, not the whole template DNA molecule (Fig. 23.01).

The components involved in the polymerase chain reaction are as follows:

1. The original DNA molecule that is to be copied is called the template and the segment of it that will actually be amplified is known as the target sequence. A trace amount of the DNA template is sufficient.

2. Two PCR primers are needed to initiate DNA synthesis. These are short pieces of single-stranded DNA that match the sequences at either end of the target DNA segment. PCR primers are made by chemical synthesis of DNA as described in Ch. 21.

polymerase chain reaction (PCR) Amplification of a DNA sequence by repeated cycles of strand separation and replication

PCR primers Short pieces of single-stranded DNA that match the sequences at either end of the target DNA segment and which are needed to initiate DNA synthesis in PCR target sequence Sequence within the original DNA template that is amplified in a PCR reaction

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FIGURE 23.02 PCR Machine or Thermocycler

(A) The thermocycler or PCR machine can be programmed to change temperature rapidly. The heat block typically changes from a high temperature such as 90°C (for denaturation) to 50°C (for primer annealing), then back to 70°C (for DNA elongation) in a matter of minutes. This may be repeated for many cycles. (B) Rows of GeneAmp PCR machines copying human DNA at the Joint Genome Institute, in Walnut Creek, California, a collaboration between three of the US Department of Energy's National Laboratories. Credit: David Parker, Science Photo Library.

Programmable control panel

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Programmable control panel

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