Analysis of Bias during PCR and Whole Genome Amplification

Mentors: Dan Aird & Andi Gnirke

The GC content of DNA is not uniform along the genome; therefore some sections of the genome are not copied as well as others during techniques such as Polymerase Chain Reaction (PCR) and Whole Genome Amplification (WGA). Bias during amplification of DNA prior to sequencing an organism’s genome will result in that genome containing regions that are under-represented or incomplete after sequencing.

With respect to PCR, Max compared the “GC bias” caused by different thermal cycler machines, and found that bias during standard PCR conditions was dependent on which thermal cycler is used. He also found that bias on fast-ramping cyclers could be slightly improved by slowing the ramp speed. In contrast, Max discovered that bias during optimized PCR conditions is not cycler-dependent. With respect to WGA, Max found that this technique also caused GC bias; however the bias appeared dependent on the GC content of the organism’s whole genome, rather than that of specific DNA regions. This bias existed regardless of the starting concentration of genomic DNA.

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Max, a senior at Cambridge Rindge & Latin School, optimized conditions to avoid amplification bias during the amplification reactions that precede DNA sequencing.