WHOLE GENOME AMPLIFICATION AND GC BIAS

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WHOLE GENOME AMPLIFICATION AND GC BIAS

Postby NIKILAYLA » Sep 08 2016 11:31 am

Hello,

I performed Multiplex PCR in WGA amplified products with SurePlex kit and in my results i found a relationship between GC content and PCR Efficiency and ADO.

The GC content of my Pcr products ranges between 26% and 50%. In my results i found that PCR products with higher GC content (50%-45%) had better PCR efficiency than pcr products with lower GC content (26%-34%).

I know from the bibliography that PCR products with high GC contend (>50%) tend to have lower percentage of PCR efficiency. I try to interpret my results , i was wondering if the annealing of primers used in SurePlex in AT rich regions is unstable and so the PCR efficiency of these regions is lower.

is this theory logic?
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Re: WHOLE GENOME AMPLIFICATION AND GC BIAS

Postby ck24 » Aug 01 2017 4:55 am

Whole genome amplification and PCR in general (for most polymerases) tend to favor GC-neutral sequences (40-50% GC). For WGA samples, you use random primers, A-T rich primers have weak interactions and are less likely to prime. GC-rich primers tend to anneal less efficiently because there are generally fewer sites available for priming due to the stronger interaction between the two strands. So WGA introduces some level bias and is well documented to have problems with evenness of coverage across the genome.

Then there's PCR itself which tends to favor amplification of GC-neutral sequences. This is generally enzyme dependent and difficult to avoid. Multiplex PCR in this step should probably only be used for QC of intact loci prior to amplification, as it is difficult to make assessments to what degree various parts of the genome are amplified without doing deep sequencing.
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