A Tale of Two Strands: Reverse-Transcriptase Polymerase Chain
Reaction Detection of Hepatitis C Virus Replication
Fig. 1. Potential causes of nonspecific amplification of
positive-strand RNA sequence by using tagged RT-PCR. (A)
Conventional reverse transcription of RNA into cDNA at 42°C
with the minus-strand specific tagged HCV primer can lead to
nonspecific synthesis of cDNA from positive-strand RNA by virtue of
(a) self-priming, (b) random prining, or (c) false priming. (B)
These nonspecific cDNAs along with some of the tagged primer will
be transferred into the subsequent PCR reaction mix. The tagged HCV
primer (through its HCV-complementary sequence) can now use the
nonspecifically amplified cDNA as template during the first cycle
of the PCR reaction. (C) In further PCR cycles, amplification of
the target by the PCR primers (tag primer, which lacks HCV
sequence, and the downstream HCV primer) leads to a false-positive
result. RNA is shown as dotted lines, and DNA as solid lines.
"Active" primer sites appear green, whereas "inactive" primer sites
are red. The tag sequence is shown in yellow.
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