A polymerase chain reaction method for the amplification of full-length envelope genes of HIV-1 from DNA samples containing single molecules of HIV-1 provirus.
Polymerase chain reaction (PCR) amplification of full-length envelope genes from the human immunodeficiency virus type 1 (HIV-1) directly from uncultured clinical samples is difficult. This paper describes a comparative assessment of the performance of three thermostable polymerases in an HIV-1 full-length envelope gene PCR. The PCR method utilising Expand HiFi polymerase was successful when using DNA samples extracted from a variety of sources including blood, semen and various tissues. This method generated high and specific yields of product from samples containing as little as one copy of HIV-1 proviral DNA. The resulting PCR products were suitable for a variety of downstream analytical methods including DNA sequence analysis.
Division of Microbiology and Infectious Diseases, University of Nottingham, Queen's Medical Centre, UK.
P
P
McClure
R
R
Curran
S
S
Boneham
J K
JK
Ball
D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005784:Gene Amplification; D015752:Genes, env / Q000235:genetics*; D019302:HIV Envelope Protein gp160; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase / Q000378:metabolism
D004279:DNA, Viral; D019302:HIV Envelope Protein gp160; C072146:Pfu DNA polymerase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase