The primers are the critical component of the PCR because primers determine the specificity of the PCR. Primers are analogous to the probes in blotting and hybridization procedures (see Chapter 6). Primers are chemically manufactured on a DNA synthesizer.
Primers are designed to contain sequences homologous to sites flanking the region to be analyzed. Primer design is therefore a critical aspect of the PCR. Primers are single-stranded DNA fragments, usually 20-30 bases in length. The forward primer must bind to the target DNA sequence just 5' to the sequences intended to be amplified. The reverse primer must bind just 5' to the sequence to be amplified on the opposite strand of the DNA. Thus, the design of primers requires some knowledge of the tar get sequence. The placement of the primers will also dictate the size of the amplified product.
Binding of primers is subject to the same physical limitations as probe binding. The primer sequence (% GC) and length affect the optimal conditions in which the primer will bind to its target. The approximate melting temperature, or Tm, of the primers can be calculated using the equation for short DNA fragments described in Chapter 6. The primer Tm can serve as a starting point for
■ Figure 7-6 After the third step (extension) of the second cycle, there are four copies of the target region.
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