High-performance liquid chromatography for routine analysis of hepatitis C virus cDNA/PCR products.
Ion-pair reversed-phase high-performance liquid chromatography on alkylated nonporous polystyrene-divinylbenzene particles with a mean diameter of 2.1 microns was used to analyze PCR products according to their chain length within a few minutes. The simple and reliable procedure allows the simultaneous separation and isolation of DNA fragments differing in chain length by 1%-5% up to a size of 500 base pairs with recovery rates exceeding 97%. A greater than 70-fold increase in sensitivity could be achieved through the use of a fluorescein-labeled primer, which allowed the determination of a 127-bp hepatitis C virus cDNA/PCR product with a lower mass detection limit of 2 fmol. Calibration curves showed excellent linearity over a range of at least 4 magnitudes. Finally, the stationary phase allowed the routine analysis of hundreds of PCR products with high reproducibility of both retention times and peak areas.
Oefner
P J
PJ
University of Linz, Austria.
Huber
C G
CG
Puchhammer-Stöckl
E
E
Umlauft
F
F
Grünewald
K
K
Bonn
G K
GK
Kunz
C
C
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000032:analysis*; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis*
D018076:DNA, Complementary; D012367:RNA, Viral