AEGiS-06IAC: Direct detection of HIV sequences in blood: high-gain polymerase chain reaction.

6th International AIDS Conference

San Francisco, California, USA — June 20-23, 1990

Direct detection of HIV sequences in blood: high-gain polymerase chain reaction.

Int Conf AIDS 1990 Jun 20-23; 6:318 (abstract no. 1019)
Faloona F, Weiss S, Ferre F, Mullis K; Department of Infectious Diseases, Cetus Corporation, Emeryville, CA, USA

OBJECTIVE: The synthesis of non-specific DNA in the polymerase chain reaction (PCR) compromises the overall yield of the specific PCR product. The detection of the low levels of HIV-1 proviral DNA in peripheral blood requires optimal sensitivity. Procedural modifications were examined to accomplish greater specificity of the PCR primer extension step.

METHODS: Varying amounts of plasmid DNA containing the HIV-1 proviral genome (eg. 1-100 copies) were amplified with primers to the gag region, in the presence of increasing amounts of human cellular DNA (eg. 0.01-10 mug). All of the reagents, except the Taq DNA polymerase, were added to the reactions at room temperature. Amplifications were then initiated by adding the enzyme at either room temperature, as routinely performed, or at temperatures above the annealing temperature of the cycling reaction.

RESULTS: Reactions were analyzed by ethidium bromide stained gel electrophoresis. 1-10 copies of HIV-1 DNA in the presence of 10 mug of human DNA can be routinely detected only when initiating the reactions by adding the DNA polymerase at elevated temperatures. The implications of this procedural modification for the detection of proviral DNA containing variant sequences at the primer binding regions will be discussed.

CONCLUSION: Highly complex DNA contains many regions capable of hybridizing with some PCR primers thereby providing a substrate for Taq DNA polymerase extension at low temperatures. Considerable primer-dependent synthesis of DNA occurs before the initial denaturation step of the PCR. Greater specificity of primer extension and therefore "higher gain" PCR is accomplished by adding the Taq DNA polymerase at elevated temperatures.

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