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Chapter 11. qPCR on LM-PCR
Amplified Samples
Chapter 11 describes the qPCR assays used to estimate relative fold-enrichment.
These assays are an inexpensive way to determine whether the capture was
successful prior to sequencing.
A standardized set of qPCR SYBR Green assays are employed as internal quality
controls for NimbleGen Sequence Capture experiments performed with human
total gDNA. The genomic loci recognized by these assays are included as capture
targets on every NimbleGen Human Sequence Capture array. Comparison by
qPCR of the relative DNA concentrations of these control loci in non-captured
and captured samples allows for the estimation of enrichment of a capture target
before committing sample libraries to expensive and/or time-consuming
downstream applications.
The internal control region assays recommended in this chapter were selected
due to their convenience for use across different species and because they
produce consistent results. It is worth considering designing and evaluating locus
specific qPCR assays for your own region(s) of interest because capture results
can vary from locus to locus. It is possible that your regions of interest may
enrich differently than our internal control regions.
For more information regarding general PCR and qPCR methods, consult the
PCR Applications Manual (3rd Edition), available from the Roche Applied
Science website (www.roche-applied-science.com).