Simultaneous assay of DNA and RNA targets in the whole blood using novel isolation procedure and molecular colony amplification.
A universal procedure that permits the whole human blood to be tested for the presence of single molecules of DNA and RNA targets is described. The procedure includes a novel protocol for the isolation of total nucleic acids from the guanidinium thiocyanate lysate of unfractionated blood in which, prior to phenol/chloroform extraction, the sample is deproteinized by precipitation with isopropanol. The procedure results in a nearly 100% yield of DNA and RNA, preserves the integrity of RNA, and removes any polymerase chain reaction (PCR) inhibitors. Following reverse transcription (RT), target molecules are counted after having been amplified as molecular colonies by carrying out PCR in a polyacrylamide gel. The entire procedure was checked by assaying viral DNA and RNA in 100-microl aliquots of the whole blood and was found to be capable of detecting 100% molecules of DNA target and 50% molecules of RNA target. Unexpectedly, nucleic acids at relatively high concentrations (1 ng/microl) were found to selectively inhibit the RT activity of Thermus thermophilus DNA polymerase without affecting its DNA-dependent polymerization activity. It follows that the popular single-enzyme RT-PCR format, in which this DNA polymerase serves for both RT and PCR, is not appropriate for assaying rare RNA targets.
Chetverina
Helena V
HV
Institute of Protein Research of the Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Falaleeva
Marina V
MV
Chetverin
Alexander B
AB
D004247:DNA / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification*; D048348:Reverse Transcription / Q000235:genetics; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000235:genetics
D012313:RNA; D004247:DNA