RNA: a method to specifically inhibit PCR amplification of known members of a multigene family b

Abstract:

The polymerase chain reaction (PCR) is a versatile method to
amplify specific DNA with oligonucleotide primers. By designing
degenerate PCR primers based on amino acid sequences that are highly conserved
among all known gene family members, new members of a multigene
family can be identified. The inherent weakness of this approach
is that the degenerate primers will amplify previously identified, in
addition to new, family members. To specifically address this problem,
we synthesized a specific RNA for each known family member so that
it hybridized to one strand of the template, adjacent to the 3′-end of the primer, allowing the degenerate
primer to bind yet preventing extension by DNA polymerase. To test our
strategy, we used known members of the soluble, nitric oxide-sensitive
guanylyl cyclase family as our templates and degenerate primers
that discriminate this family from other guanylyl cyclases. We demonstrate
that amplification of known members of this family is effectively
and specifically inhibited by the corresponding RNAs, alone or in
combination. This robust method can be adapted to any application where
multiple PCR products are amplified, as long as the sequence of
the desired and the undesired PCR product(s) is sufficiently distinct
between the primers.

Nucleic Acids Research.2001:29(6):e31

Department of Biochemistry, University of Tennessee, 858 Madison Avenue, Suite G01, Memphis, TN 38

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