Direct amplification and cloning of up to 5-kb lentivirus genomes from serum.

Important note: Information in this article was accurate in 1997. The state of the art may have changed since the publication date.

Direct amplification and cloning of up to 5-kb lentivirus genomes from serum.

Biotechniques. 1996 Aug;21(2):312-9. Unique Identifier : AIDSLINE MED/97016181
Holterman L; Mullins JI; Haaijman JJ; Heeney JL; Biomedical Primate Research Centre, Rijswijk, The Netherlands.

Abstract: To produce large cDNA strands from biological samples containing limited numbers of template molecules, it may be necessary to minimize both nonspecific primer attachment in first-strand synthesis and secondary structure in RNA molecules. Failure to do so could result in the accumulation of shortened cDNA strands and therefore may reduce the yield of large cDNA molecules, sometimes below detection level. We show that 5.0-kb cDNA fragments can be generated from simian immunodeficiency virus RNA in a specific reverse transcription (RT)-PCR by increasing the stringency of the primer-annealing conditions, followed by the elimination of excess free primer. Since this method utilizes a relatively long primer in the first-strand cDNA synthesis, it is possible to heat-denature the nonspecific RNA/primer complexes and RNA secondary structure without dissociating the primer from the specific template. In contrast to classic RT assays, in which an excess of primer is annealed to denatured RNA just prior to and during reverse transcription at relative low temperatures (37 degrees-42 degrees C), this method eliminates false priming. To optimize the yield and fidelity of full-length cDNA molecules, two PCR amplifications are first performed using both Taq and Pfu polymerase, followed by Pfu alone in the second amplification.

Keywords: *Cloning, Molecular/METHODS *DNA, Complementary/GENETICS *Polymerase Chain Reaction/METHODS *RNA, Viral/BLOOD *SIV/GENETICS
970630
M9761241

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