[CITATION][C] Reverse transcriptase can inhibit PCR and stimulate primer-dimer formation.

KM Chumakov - Genome Research, 1994 - genome.cshlp.org
KM Chumakov
Genome Research, 1994genome.cshlp.org
Presence of residual reverse transcriptase (RT) activity in cDNA used for PCR results in
accumulation of primer-dimers and in inhibition of amplification of a specific DNA product.
Inactivation of RT by heat or some other method solves both these problems. We suggest
that RT is capable of adding nucleotides at the 3'end of primers before PCR amplification
begins, thereby extending short complementary regions in primers facilitating their
interaction. RT should be inactivated in all protocols involving PCR amplification of cDNA …
Presence of residual reverse transcriptase (RT) activity in cDNA used for PCR results in accumulation of primer-dimers and in inhibition of amplification of a specific DNA product. Inactivation of RT by heat or some other method solves both these problems. We suggest that RT is capable of adding nucleotides at the 3'end of primers before PCR amplification begins, thereby extending short complementary regions in primers facilitating their interaction. RT should be inactivated in all protocols involving PCR amplification of cDNA. PCR amplification of cDNA synthesized in vitro by reverse transcriptase (RT-PCR) is used widely in studies of both cellular and viral RNAs, enabling the powerful tools of DNA research to be used for studies of RNA.(1~ One of the applications of RT-PCR is to detect and quantitate neurovirulent revertants in oral poliovirus vaccine (OPV).(2'3~ Mutant analysis by PCR and restriction enzyme cleavage (MAPREC), developed in our laboratory for this purpose, involves synthesis of poliovirus cDNA by RT, and amplification of the DNA segment under study by PCR with simultaneous creation of restriction sites that are affected by the mutation of interest. While using this method for quantitation of numerous mutations in three types of OPV, we found that some crude cDNA preparations (ie, products of RT reactions without any further treatment) contained inhibitor (s) that blocked PCR amplification of DNA. Dilution of such cDNA samples restored PCR amplification. Another problem with crude cDNA samples was the apparent enhancement of" the formation of artifactual DNA products, presumably primer-dimers. In this short communication, we report that both of these problems are caused by RT itself and not by any other component of the RT reaction mixture or the RT storage buffer. Phenol extraction or heating of newly synthesized cDNA solved both of these problems. Incubation of primers with RT and dNTPs resulted in the incorporation of [o~-32p] dATP into primer molecules, suggesting that enzymatic extension of primer molecules may be responsible for their increased tendency to form dimers and inhibition of amplification of specific DNA products.
genome.cshlp.org