hard · MCAT bio-biochem
A researcher performs PCR using a pair of primers flanking a target sequence, then runs the product on native polyacrylamide gel electrophoresis alongside a size ladder. Unexpectedly, the amplicon migrates as a smear spanning a broader-than-expected size range, and melting curve analysis reveals two distinct melting temperatures (T_m) for the product population.
Which explanation best accounts for both observations?
- The Taq polymerase used lacks 3' to 5' exonuclease proofreading activity, so it introduces random single-nucleotide substitutions during synthesis.
- The template contains a heterozygous VNTR between the primer sites, generating two allele-specific amplicon lengths.
- The annealing temperature was set too low, causing nonspecific primer binding throughout the genome.
- The PCR reaction ran for too many cycles, exhausting the dNTP pool before completion.
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