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 cri tical controls for genes lacking introns. The convenient approaches of either designing oligonucleotide primers at the splice junction or di fferentiating the target sequence based on the size difference obtaine d 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 s trand separation temperature requirements for PCR amplification, Follo wing reverse transcription of the RNA by recombinant Thermus thermophi lus DNA polymerase (rTth pol), the resulting RNA DNA hybrid is digeste d by the RNase H activity of rTth pol, allowing the PCR primer to hybr idize and initiate second-strand cDNA synthesis, Substitution of one o r more conventional nucleotides with nucleotide analogs that decrease base stacking interactions and/or hydrogen bonding (e.g, hydroxymethyl dUTP or dITP) during the first- and second-strand cDNA synthesis step reduces the strand separation temperature of the resultant DNA DNA dup lex, Alteration of the thermal cycling parameters of the subsequent PC R amplification, such that the strand separation temperature is below that required for denaturation of genomic duplex DNA composed of stand ard nucleotides, prevents the genomic DNA from being denatured and the refore amplified.