Enzymatic amplification of RNA purified from crude cell lysate by reversible target capture.
For increased clinical applications of nucleic acid probes to gene diagnosis, current procedures must be modified to become more amenable to the rapid processing of many samples with as few manipulations as possible. Here we summarize progress in the development of a strategy for performing molecular hybridization directly in lysate of biological samples dissolved in solutions containing the chaotropic agent, guanidine thiocyanate. Hybrids are purified by a process referred to as "reversible target capture," in which specific nucleic acid sequences are rapidly purified from crude lysate. We illustrate the use of this strategy to assay HIV-1 RNA and to rapidly purify HIV-1 RNA before enzymatic amplification by the polymerase chain reaction method.
Thompson
J D
JD
Department of Neoplastic Diseases, Hahnemann University, Philadelphia, PA 19102.
Decker
S
S
Haines
D
D
Collins
R S
RS
Feild
M
M
Gillespie
D
D
D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis*; D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D006146:Guanidines; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D015336:Molecular Probe Techniques; D011070:Poly I-C; D011072:Poly U; D011131:Polyribonucleotides / Q000494:pharmacology; D012333:RNA, Messenger / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification*; D013861:Thiocyanates
D000998:Antiviral Agents; D006146:Guanidines; D011131:Polyribonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D013861:Thiocyanates; D011072:Poly U; C054436:guanidine thiocyanate; C047490:poly(I).poly(c12,U); D004259:DNA-Directed DNA Polymerase; D011070:Poly I-C