Polymerase chain reaction fidelity and denaturing high-performance liquid chromatography.
Incorporation of non-complementary nucleotides during polymerase chain reaction can result in ambiguous denaturing high-performance liquid chromatography profiles that reduce both sensitivity and specificity of mutation analysis. The use of proofreading DNA polymerases increases the fidelity of polymerase chain reaction and, consequently, reduces background noise in the chromatograms. This is demonstrated for several BRCA1 and BRCA2 mutations hat had yielded previously chromatograms of poor quality using non-proofreading enzyme for amplification. Interestingly, despite the reduced level of background heteroduplices, the ability of denaturing high-performance liquid chromatography to detect mutant alleles at a frequency <10% in pools of chromosomes did not improve significantly.
Muhr
Daniela
D
Department of Obstetrics and Gynecology, Division of Senology, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
Wagner
Teresa
T
Oefner
Peter J
PJ
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017931:DNA Primers; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity
D017931:DNA Primers