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DNA Amplification for Forensic Analysts


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Primer concentrations between 0.1 and 0.5µM are generally used. This relatively high concentration compared to the DNA template helps to drive the PCR reaction. Higher primer concentrations may promote mispriming and accumulation of nonspecific product and may increase the probability of generating primer-dimer template-independent artifacts. Because nonspecific products and primer-dimer artifacts are substrates for PCR, they compete with the desired product for enzyme, dNTPs, and primers, resulting in a lower yield.

Mutations in the primer binding site region can result in null alleles. When common primer binding site mutations are identified, it is common practice to use degenerate primers for these loci. Degenerate primers are a set of primers that have a number of options at several positions in the sequence, which allows annealing to and amplification of a variety of related sequences. The use of degenerate primers can reduce the specificity of the primer(s), increasing both the likelihood of mismatches and the level of background noise. However, use of degenerate primers allows the amplification of those null alleles without significantly altering the overall performance of the amplification.

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