Pcr Components

A PCR reaction is prepared by mixing several individual components and then adding deionized water to achieve the desired volume and concentration of each of the components. Commercial kits with pre-mixed components may also be used for PCR. These kits have greatly simplified the use of PCR in forensic DNA laboratories.

The most important components of a PCR reaction are the two primers, which are short DNA sequences that precede or 'flank' the region to be copied. A primer acts to identify or 'target' the portion of the DNA template to be copied. It is a chemically synthesized oligonucleotide that is added in a high concentration relative to the DNA template to drive the PCR reaction.

Table 4.1

Number of target DNA molecules created by PCR amplification (if reaction is working at 100% efficiency). The target PCR product is not completely defined by the forward and reverse primers until the third cycle.

Cycle Number

Number of Double-stranded Target

Molecules (Specific PCR Product)

Table 4.1

Number of target DNA molecules created by PCR amplification (if reaction is working at 100% efficiency). The target PCR product is not completely defined by the forward and reverse primers until the third cycle.

1

G

2

G

3

2

4

4

E

8

6

16

7

32

8

64

9

128

1G

2E6

11

E12

12

1G24

13

2G48

14

4G96

1E

8192

16

16 384

17

32 768

18

6EE36

19

131 G72

2G

262144

21

E24288

22

1 G48E76

23

2G971E2

24

41943G4

2E

8 388 6G8

26

16777216

27

33 EE4432

28

67 1G8864

29

134217728

3G

26843E4E6

31

E3687G912

32

1G73 741 824

Some knowledge of the DNA sequence to be copied is required in order to select appropriate primer sequences.

The other components of a PCR reaction consist of template DNA that will be copied, building blocks made up of each of the four nucleotides, and a DNA polymerase that adds the building blocks in the proper order based on the template DNA sequence. The various components and their optimal concentrations are listed in Table 4.2. Thermal stable polymerases that do not fall apart during the near-boiling denaturation temperature steps have been important to the success of PCR (Saiki et al. 1988). The most commonly used thermal stable polymerase is Taq, which comes from a bacterium named Thermus aquati-cus that inhabits hot springs.

When setting up a set of samples that contain the same primers and reaction components, it is common to prepare a 'master mix' that can then be dispensed in equal quantities to each PCR tube. This procedure helps to insure that there is more homogeneity between samples. Also by setting up a larger number of reactions at once, small pipetting volumes can be avoided, which improves the accuracy of adding each component (and thus the reproducibility of one's method). When performing a common test on a number of different samples, the goal should be to examine the variation in the DNA samples not variability in the reaction components used and the sample preparation method.

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