An assessment of optimal conditions for amplification of HIV cDNA using Thermus aquaticus polymerase.
Optimal conditions for in-vitro enzymatic DNA amplification using Thermus aquaticus polymerase were defined using env and gag sequences of human immunodeficiency virus type I as templates. It was found that specific amplification of the target sequence occurred when the primer annealing temperature was above 55 degrees C. Polymerase added once at the beginning of the procedure was sufficient for good results. Deoxynucleotide and primer concentrations and chain elongation time were not found to be important as long as they were kept above a critical level. Differences were noted between the efficiency of specific amplification from purified plasmid and genomic DNA. A rapid and simple method without the use of radioactive reagents for confirmation of a suggestive positive result on gel electrophoresis using restriction enzyme digestion of the amplified products is described. Some possible drawbacks of the technique particularly if used as a diagnostic tool are discussed.
Carman
W F
WF
National Institute for Virology, University of the Witwatersrand, Johannesburg, South Africa.
Kidd
A H
AH
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D015497:HIV-1 / Q000235:genetics*; D015151:Immunoblotting; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D019914:Taq Polymerase; D013696:Temperature; D013698:Templates, Genetic; D013824:Thermus / Q000201:enzymology; D013997:Time Factors; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
D004279:DNA, Viral; D014764:Viral Proteins; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes