Polymerase chain reaction (PCR) has been reported as one of the most efficient techniques to detect hepatitis C virus (HCV) RNA. The determination of the specificity of PCR products is usually based on 'nested' PCR, Southern blotting and hybridization of the amplified DNA to radioactive oligonucleotide probes recognizing sequences comprised between the PCR primers. The recent introduction of capillary electrophoresis (CE) to analyse DNA fragments and PCR products appears to be very interesting because this technology is rapid, reproducible, sensitive and could be suitable to detect DNA/DNA and DNA/RNA hybrids. We demonstrate that specific hybridization of an HCV oligonucleotide probe to single stranded HCV-DNA obtained by unbalanced PCR is detectable by capillary electrophoresis, therefore enabling a one-step, non-radioactive protocol to demonstrate the specificity of amplification of HCV sequences by PCR
Detection of hepatitis C virus by unbalanced polymerase-chain reaction, hybridization to synthetic oligonucleotides and capillary electrophoresis
BIANCHI, Nicoletta;MISCHIATI, Carlo;FERIOTTO, Giordana;GAMBARI, Roberto
1994
Abstract
Polymerase chain reaction (PCR) has been reported as one of the most efficient techniques to detect hepatitis C virus (HCV) RNA. The determination of the specificity of PCR products is usually based on 'nested' PCR, Southern blotting and hybridization of the amplified DNA to radioactive oligonucleotide probes recognizing sequences comprised between the PCR primers. The recent introduction of capillary electrophoresis (CE) to analyse DNA fragments and PCR products appears to be very interesting because this technology is rapid, reproducible, sensitive and could be suitable to detect DNA/DNA and DNA/RNA hybrids. We demonstrate that specific hybridization of an HCV oligonucleotide probe to single stranded HCV-DNA obtained by unbalanced PCR is detectable by capillary electrophoresis, therefore enabling a one-step, non-radioactive protocol to demonstrate the specificity of amplification of HCV sequences by PCRI documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
