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Mid-length strands are synthesized from the full-length templates. The sites to which the primers annealed dictate the 5' end of these strands.

When the mid-length strands are used as templates, short strands are generated.

When the short strands are used as templates, like-sized strands are generated.

Selecting Primer Pairs

The nucleotide sequences of the two primers are critical because it is the primers that dictate which portion of the DNA is amplified. Each must be complementary to a sequence on the appropriate strand, flanking the region to be amplified. The synthesis reaction will then add nucleotides onto the primers, elongating the DNA chain so that the target DNA between those primers is copied. Thus, if a researcher wants to amplify a DNA sequence that encodes a specific protein, then the researcher must first determine the nucleotide sequences flanking the gene that encodes the protein and then synthesize the appropriate pair of oligonucleotides to serve as primers.

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