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Abstract
Restriction endonuclease digestion was used to eliminate false-positive signals caused
by polymerase chain reaction (PCR) product DNA contamination in a reverse transcribed
(RT) PCR for amplifying rubella virus (RV) RNA sequences. A restriction enzyme selected
to cut the PCR product DNA between, but not within, the primer binding sites was used
to digest reaction mixtures after reverse transcription but before PCR amplification.
Because restriction enzymes generally react only with specific double-strand sequences,
contaminating DNA was rendered inactive while reverse-transcribed single strand cDNA
was amplified. Assays showed that restriction enzyme digestion reduced template activity
of product DNA by a factor of 10(7), while leaving sensitivity of the RT-PCR unaffected.