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

Nucleic Acids Res. 1998 Apr 1;26(7):1628-35. doi: 10.1093/nar/26.7.1628.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arthritis, Rheumatoid / genetics
  • Base Sequence
  • Codon
  • Computer Communication Networks
  • Consensus Sequence
  • Conserved Sequence
  • DNA Modification Methylases / chemistry*
  • DNA Modification Methylases / genetics
  • DNA Primers*
  • Evolution, Molecular*
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Hybridization
  • Phylogeny*
  • Polymerase Chain Reaction / methods
  • RNA-Directed DNA Polymerase / chemistry*
  • RNA-Directed DNA Polymerase / genetics
  • Sarcoma, Kaposi / genetics
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Software

Substances

  • Codon
  • DNA Primers
  • DNA Modification Methylases
  • RNA-Directed DNA Polymerase