Primer extension analysis provides a sensitive tool for the identification of PCR-amplified DNA from HIV-1.
Primer extension analysis was evaluated as a means to identify PCR-amplified DNA from HIV-1. Solution hybridization with radioactive labeled oligonucleotides followed by an extension reaction with Klenow fragment of Escherichia coli DNA polymerase I and subsequent separation on denaturing polyacrylamide gels reveals single stranded DNA products of the predicted size. The specificity of the reaction is further demonstrated by specific endonuclease digestion. The analysis is more sensitive than Southern blotting and about equally sensitive as Slot blot analysis when double-stranded DNA probes are used for hybridization. With end-labeled oligonucleotide probes, primer extension analysis proved an order of magnitude more sensitive than membrane hybridization. The analysis also allows quantitation of amplified DNA from 1 pg to about 1 ng of DNA product. Under the conditions described for amplification, primer extension analysis is capable of detecting a single HIV-1 plasmid DNA molecule in the presence of 1 microgram of total DNA. 3'-end mismatching of the oligonucleotide probe does not result in a significantly altered detection limit. Primer extension analysis can also be carried out with at least three different PCR-amplified DNAs in the same reaction tube.
Swiss National Center for Retroviruses, University of Zurich.
J
J
Böni
J
J
Schüpbach
D001483:Base Sequence; D015139:Blotting, Southern; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D004279:DNA, Viral