Selective amplification of RNA utilizing the nucleotide analog dITP and Thermus thermophilus DNA polymerase.
The ability to selectively amplify RNA in the presence of genomic DNA of analogous sequence is cumbersome and requires implementation of critical controls for genes lacking introns. The convenient approaches of either designing oligonucleotide primers at the splice junction or differentiating the target sequence based on the size difference obtained by the presence of the intron are not possible. Our strategy for the selective amplification of RNA targets is based on the enzymology of a single thermostable DNA polymerase and the ability to modulate the strand separation temperature requirements for PCR amplification. Following reverse transcription of the RNA by recombinant Thermus thermophilus DNA polymerase (rTth pol), the resulting RNAxDNA hybrid is digested by the RNase H activity of rTth pol, allowing the PCR primer to hybridize and initiate second-strand cDNA synthesis. Substitution of one or more conventional nucleotides with nucleotide analogs that decrease base stacking interactions and/or hydrogen bonding (e.g. hydroxymethyldUTP or dITP) during the first- and second-strand cDNA synthesis step reduces the strand separation temperature of the resultant DNAxDNA duplex. Alteration of the thermal cycling parameters of the subsequent PCR amplification, such that the strand separation temperature is below that required for denaturation of genomic duplex DNA composed of standard nucleotides, prevents the genomic DNA from being denatured and therefore amplified.
Auer
T
T
Program in Core Research, Roche Molecular Systems, Alameda, CA 94501, USA.
Sninsky
J J
JJ
Gelfand
D H
DH
Myers
T W
TW
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D007293:Inosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology*
D012329:RNA, Bacterial; D011994:Recombinant Proteins; D007293:Inosine Triphosphate; C065360:2'-deoxyinosine triphosphate; C093010:DNA polymerase, Thermus thermophilus; D004259:DNA-Directed DNA Polymerase