RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers.
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.
Department of Biochemistry, University of Tennessee, 858 Madison Avenue, Suite G01, Memphis, TN 38163, USA. pyuen@utmem.edu
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Yuen
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Brooks
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D000818:Animals; D017931:DNA Primers / Q000235:genetics*; D016373:DNA, Antisense / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006162:Guanylate Cyclase / Q000235:genetics; D005810:Multigene Family / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D021122:Protein Subunits; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D013698:Templates, Genetic
D017931:DNA Primers; D016373:DNA, Antisense; D018076:DNA, Complementary; D021122:Protein Subunits; D012313:RNA; D006162:Guanylate Cyclase