Elimination of locus-specific inter-individual variation in quantitative PCR.
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 degrees 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.
Shi
Jinxiu
J
City of Hope National Medical Center, Duarte, CA 91010-3000, USA.
Liu
Qiang
Q
Nguyen
Vu Q
VQ
Sommer
Steve S
SS
D016477:Artifacts; D004247:DNA / Q000032:analysis* / Q000235:genetics* / Q000528:radiation effects; D004252:DNA Mutational Analysis / Q000379:methods*; D017353:Gene Deletion; D020440:Gene Duplication; D020869:Gene Expression Profiling / Q000379:methods; D014644:Genetic Variation / Q000235:genetics*; D006358:Hot Temperature; D016133:Polymerase Chain Reaction / Q000379:methods*; D040641:Quantitative Trait Loci / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods
D004247:DNA