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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.
The PCR process was originally developed to amplify short segments of a longer DNA molecule (Saiki et al. 1985). A typical amplification reaction includes target DNA, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium.f an amplification reaction fails and you suspect the DNA template is contaminated with an inhibitor, add the suspect DNA preparation to a control reaction with a DNA template and primer pair that has amplified well in the past.