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Nucleic Acids Res. 1998 Apr 1;26(7):1628-35.

Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences.

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  • 1Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, WA 98195, USA.

Abstract

We describe a new primer design strategy for PCR amplification of unknown targets that are related to multiply-aligned protein sequences. Each primer consists of a short 3' degenerate core region and a longer 5' consensus clamp region. Only 3-4 highly conserved amino acid residues are necessary for design of the core, which is stabilized by the clamp during annealing to template molecules. During later rounds of amplification, the non-degenerate clamp permits stable annealing to product molecules. We demonstrate the practical utility of this hybrid primer method by detection of diverse reverse transcriptase-like genes in a human genome, and by detection of C5DNA methyltransferase homologs in various plant DNAs. In each case, amplified products were sufficiently pure to be cloned without gel fractionation. This COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy has been implemented as a computer program that is accessible over the World Wide Web (http://blocks.fhcrc.org/codehop.html) and is directly linked from the BlockMaker multiple sequence alignment site for hybrid primer prediction beginning with a set of related protein sequences.

PMID:
9512532
[PubMed - indexed for MEDLINE]
PMCID:
PMC147464
Free PMC Article
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