Optimisation and sensitivity of single-stranded conformation polymorphism for the detection of hepatitis C virus quasi-species.
Single-stranded conformation polymorphism (SSCP) is a technique used widely for the detection of differences in DNA sequence based on PCR technology. Developed by geneticists for the detection of mutations causing disease, it has been adopted more recently for the analysis of the quasi-species of viral genomes, such as hepatitis C virus (HCV). The rigorous standardisation and determination of the limit of detection of the technique has rarely been shown. Variants within the quasi-species of the hypervariable region of HCV were cloned into pUC119 and the resulting plasmids quantitated and used as templates to optimise SSCP. Variables studied included the number of variants detected, the sensitivity of detection of variants in the minority, the electrophoresis temperature, methods of generation of single-stranded DNA, effect of numbers of cycles of PCR and use of DNA polymerase with proof-reading ability. It was demonstrated that the optimised method could detect at least five variants within a quasi-species and that variants could be detected down to a level of 2% of the quasi-species. Electrophoresis at room temperature for 18 h was highly reproducible. Generation of single-stranded DNA using a single primer with Taq polymerase for 20 cycles gave an accurate reflection of the quasi-species make-up and use of Pfu polymerase reduced sensitivity of detection of minor bands. This SSCP method provides an accurate tool to evaluate HCV quasi-species.
Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, G11 5JR, Glasgow, UK.
V M
VM
McKechnie
E A
EA
McCruden
D001483:Base Sequence; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000235:genetics*; D004279:DNA, Viral / Q000097:blood; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D014644:Genetic Variation; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D019698:Hepatitis C, Chronic / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D022801:Complementarity Determining Regions; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase