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Primer sequences are determined from known sequence since there must be a match to the region of DNA to be amplified. PCR amplification requires 2 primers that determine the region of sequence amplified in the forward and reverse direction.ikewise, the reverse primer is designed from the complimentary strand. PCR is exponential amplification in which the newly generated PCR fragment from one cycle also acts as a template for the next cycle (Figure 1A). Amplification of DNA for Sanger sequencing differs from PCR in that a single primer is used.
In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. 2  The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).