Elimination of locus-specific inter-individual variation in quantitative PCR

摘要

Robust dosage PCR (RD-PCR), a duplex and quantitative PCR, detects large heterozygous deletions and duplications, in genomic DNA samples. RD-PCR amplifies an endogenous internal control and a target locus. Two of six RD-PCR assays behaved anomalouslydue to lower yields specific to the targets. The variability was eliminated by heat treatment of the genomic DNA samples in 2x TE solution at 90°C for 10 min. Heat treatment improves the utility of RD-PCR and may be generally helpful in multiplex. PCR quantitation. The mechanism by which heat treatment eliminates inter-individual variation is unclear: The variability is not associated with DNA extraction methods, RNA contamination or solution protein contamination but may reflect inhibition from tightly bound chromatin proteins.