The PCR process

The PCR amplifies specific regions of template DNA. The power of the technique is illustrated in Table 5.1 In theory, a single molecule can be amplified one billion-fold by 30 cycles of amplification; in practice the PCR is not 100 % efficient but does still produce tens of millions of copies of the target sequence [19].

The amplification of DNA occurs in the cycling phase of PCR, which consists of three stages (Figure 5.2): denaturation, annealing and extension. In the denaturation stage the reaction is heated to 94 °C; this causes the double stranded DNA molecule to 'melt' forming two single stranded molecules. DNA melts at this temperature because the hydrogen bonds that hold the two strands of the DNA molecule together are relatively weak. As the temperature is lowered, typically to between 50 and 65 °C, the oligonucleotide primers anneal to the template. The primers are in molar excess to the template strands and bind to the complementary sequences before the template DNA reassociates to form double stranded DNA. After the primers have annealed the temperature is increased to 72 °C, which is in the optimum temperature range for the Taq polymerase. Nucleotides are added to the nascent DNA strand at the rate of approximately 40-60 per second [26, 27]. The enzyme catalyses the addition of nucleotides to the 3' ends of the primers using the original DNA strand as a template; it has a high pro-cessivity, catalysing the addition of approximately 50 nucleotides to the nascent DNA strand before the enzyme dissociates - several Taq polymerase enzymes will associate and disassociate during the extension phase of longer PCR products.

The normal range of cycles for a PCR is between 28 and 32. In extreme cases, where the amount of target DNA is very low the cycle number can be increased to up to 34 cycles. It has been demonstrated that going above this cycle number does

Table 5.1 The PCR can theoretically multiply DNA over 1 billion-told after 32 cycles - in reality it is not 100 % efficient but is still extremely powerful. (The AmpRSTR® and PowerPlex® STR kits are described in Chapter 6)

Number of

Cycle

PCR products

1

0

2

0

3

2

4

4

5

8

6

16

7

32

8

64

9

128

10

256

20

262 144

28

67108 864

30

268 435 456

32

1 073 741 824

34

Standard cycle number using the AmpR STR® SGM Plus® and Identifier® kits

Standard cycle number using the PowerPlex® 16 kit Maximum number of cycles normally used in forensic analysis

Double stranded DNA

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT1 template molecule ; i i i i i i i i i i i i i i— - hydrogen bonds hold the two strands together

The temperature is increased to 94 °C. This causes the hydrogen bonds to break and results in two denatured single stranded DNA molecules.

Two single stranded DNA molecules

The temperature is reduced to 50-65 °C allowing primers to anneal to complementary sequences. The two primers must anneal to the two different strands and must be extended towards each other.

The temperature is increased to 72 °C. The enzyme Taq polymerase finds the free ends of the primers (indicated by the arrow heads) and starts to incorporate nucleotides that are complementary to the template strand.

Hydrogen bonds stabilize the template-primer interaction. The arrow head indicates the direction of primer extension

The end product is two double stranded copies of the template DNA.

Figure 5.2 The PCR process - each PCR cycle consists of three phases: denaturing, annealing and extension

1 Hold 94.0

11:00

72.0

1:00

1:00

2 Holds

Method: SGM+

Figure 5.3 A typical PCR programme involves three phases: a pre-incubation at 94 °C, which activates the AmpliTaq Gold® polymerase; the cycling phase; and a terminal incubation that maximizes the non-template addition at the end of the amplification not increase the likelihood of obtaining a profile but does increase the probability of artefacts forming during the PCR [28]. Using 34 cycles is known as low copy number (LCN) PCR and it is sparingly employed as extreme precautions have to be taken to reduce the chance of contamination - the more cycles the higher the chance of detecting contaminating DNA. The interpretation of the profiles generated using a high cycle number also become more complex.

Following the cycling phase the reaction is incubated between 60 and 72 °C for up to one hour. In addition to the template-dependent synthesis of DNA the Taq polymerase also adds an additional residue to the 3' end of extended DNA molecule, this is nontemplate dependent; the incubation at the end of the reaction is to ensure that the non-template addition is complete. The conditions of a typical PCR are shown below in Figure 5.3.

The PCR requires tightly controlled thermal conditions and these are achieved by using a thermocycler. This consists of a conducting metal block that contains heating and cooling elements with wells that accommodate the plastic reaction tubes. The temperature of the PCR block is controlled by a small microprocessor. Most thermocyclers also contain a lid that is heated to over 100 °C; this prevents the reaction evaporating and condensing on the cooler lid and thereby maintains the reaction volume, thus keeping the concentration of the reaction components stable throughout the PCR.

After amplification the results of a PCR can be visualized on an agarose gel. In a reaction that amplifies only one locus a single band should be detected (Figure 5.4).

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