Elimination of locus-specific inter-individual variation in quantitative PCR
Shi, J.; Liu, Q.; Nguyen, V.Q.; Sommer, S.S.
Biotechniques 37(6): 934-938
2004
ISSN/ISBN: 0736-6205 PMID: 15597542 Document Number: 569278
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 anomalously due 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.