Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences.
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.
Rose
T M
TM
Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, WA 98195, USA.
Schultz
E R
ER
Henikoff
J G
JG
Pietrokovski
S
S
McCallum
C M
CM
Henikoff
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000235:genetics; D001483:Base Sequence; D003062:Codon; D003195:Computer Communication Networks; D016384:Consensus Sequence; D017124:Conserved Sequence; D015254:DNA Modification Methylases / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers; D019143:Evolution, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D012514:Sarcoma, Kaposi / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012984:Software
D003062:Codon; D017931:DNA Primers; D015254:DNA Modification Methylases; D012194:RNA-Directed DNA Polymerase