Elimination of locus-specific inter-individual variation in quantitative PCR. (December 2004)

• Record Type: Journal Article
• Authors: Shi, Jinxiu; Liu, Qiang; Nguyen, Vu Q.; Sommer, Steve S.
• Is Part Of: Biotechniques. Volume 37:Number 6(2004)
• Page Start: 934
• Page End: 938
• Publication Date: 2004-12
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/04376ST01
• Languages: English
• ISSNs: 0736-6205

Abstract

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 2× 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.