A Tale of Two Strands: Reverse-Transcriptase Polymerase Chain
Reaction Detection of Hepatitis C Virus Replication
Fig. 2. Improved specificity of rTth RP-PCR for detection
of minus-strand RNA. (A) Reverse transcription of RNA into cDNA is
performed at 70°C in the presence of Mn2+. Because of the
higher temperature, there is greatly reduced (a) self-priming, (b)
random priming, or (c) false priming. Thus, there is very little
nonspecific cDNA synthesized and transferred into the PCR reaction.
This results in 1,000- to 10,000-fold specificity for detection of
the minus-strand RNA. (B) Correct detection of minus-strand RNA. As
in (A), reverse transcription of RNA into cDNA is performed at
70°C in the presence of Mn2+. The tagged HCV primer results in
synthesis of a positive-strand cDNA. In the subsequent PCR
reaction, the reverse-transcriptase activity of the rTth polymerase
is eliminated by the addition of ethyleneglycoltetraacetic acid and
Mg2+ is added to activate the DNA polymerase activity. The
positive-strand cDNA is now amplified by the tag primer (through
its tag sequence) and the downstream HCV primer.
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