PCR Practical Considerations

Although PCR is a powerful tool, there are a number of technical considerations that are inherent in the method. These are important to note in designing experiments and in interpreting data. The three most important of these are the (1) introduction of mutations into DNA PCR products by Taq polymerase, (2) mishybridization of primers to related target sequences to generate incorrect products, and (3) contamination of starting material.

The first of these relates to a feature of the Taq polymerase enzyme. When synthesizing a new strand of DNA from a template strand, it is essential that polymerases be able to correctly incorporate the proper nucleotide that is complementary to the nucleotide of the template strand being copied. The fidelity of replication is important because misincorporation of an incorrect nucleotide may introduce a mutation that can be lethal to an organism. Many polymerases contain an exonuclease enzymatic feature that allows them to overcome this problem.

This exonuclease activity of a polymerase allows the enzyme to recognize that an incorrect base has been incorporated and to cleave the nucleotide, allowing reincorporation of the correct base. Polymerases that have this “proofreading” or exonuclease activity typically have error rates in the range of 1 in 105-106 nucleotides copied. Taq polymerase lacks this exonuclease activity and has an approximate error rate for misincorporation of a nucleotide of 1 in 104105 nucleotides copied.

This high error rate of Taq may present a problem in the interpretation of data. For example, if DNA samples from patients are being analyzed in order to determine if a gene mutation correlates with a clinical disease, it must be confirmed that nucleotide differences between the DNA of normal and that of affected patients did not arise artifactually from the PCR of samples.

To address this problem, multiple individually prepared samples can be subjected to PCR reaction and characterized. Also, other polymerases have been described that have a much greater fidelity rate than Taq. One of these, called Vent, which was isolated from a submarine thermal vent, is heat stable and contains a “proofreading” exonuclease activity, giving it a 5- to 15-fold higher fidelity rate than Taq.

The second problem associated with PCR is the hybridization of primers to incorrect regions of target DNA. Primers that are not 100% matches to their specific targets must sometimes be used when the exact sequence of the region to be amplified is not known. Also, oligonucleotide primers, which are 100% homologous to their target regions, may also share some homology with other regions of a DNA sample being analyzed.

This is especially true when the sample contains a large number of varied sequences such as that prepared from total cellular DNA. The misprinting caused by hybridization to incorrect target regions may cause the generation of false products. The ability of PCR to yield false product should be considered when interpreting data and when designing primers. Primers that form highly stable hybrids to the DNA target regions should preferentially be used.

The final problem associated with PCR is the contamination of samples being analyzed with other DNA. Because PCR is able to amplify a very small amount of material, a minimal amount of cross-contamination between samples can lead to the misinterpretation of data. An example of this is using PCR to examine patient samples to detect viral DNA sequences and to diagnose an infection.

A negative sample can be contaminated by the aeration of droplets from the handling of another viral positive patient’s sample in the same area. Because cross-contamination is such a problem, special precautions, such as aeration-filtered pipets, are often used in laboratories where specific qualitative analyses of samples are essential.

 






Date added: 2024-07-02; views: 27;


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