Consensus-degenerate Hybrid Oligonucleotide Primers Research Articles

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design.

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).

Consensus-degenerate hybrid oligonucleotide primers for amplification of priming glycosyltransferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group.

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS(-)) and EPS-producing (EPS(+)) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS(+) bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS(+) strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.

Analysis of 4.3 kilobases of divergent locus B of macaque retroperitoneal fibromatosis-associated herpesvirus reveals a close similarity in gene sequence and genome organization to Kaposi's sarcoma-associated herpesvirus.

We previously identified retroperitoneal fibromatosis-associated herpesvirus (RFHV) as a simian homolog of Kaposi's sarcoma-associated herpesvirus (KSHV) in a fibroproliferative malignancy of macaques that has similarities to Kaposi's sarcoma. In this report, we cloned 4.3 kb of divergent locus B (DL-B) flanking the DNA polymerase gene from two variants of RFHV from different species of macaque with a consensus degenerate hybrid oligonucleotide primer approach. Within the DL-B region of RFHV, viral homologs of the cellular interleukin-6, dihydrofolate reductase, and thymidylate synthase genes were identified, along with a homolog of the gammaherpesvirus open reading frame (ORF) 10. In addition, a homolog of the KSHV ORF K3, the modulator of immune recognition-1, was identified. Our data show a close similarity in sequence conservation, gene content, and genomic structure between RFHV and KSHV which strongly supports the grouping of these viral species within the same RV-1 rhadinovirus lineage and the hypothesis that RFHV is the macaque homolog of KSHV.

Use of degenerate primers and touchdown PCR for construction of cDNA libraries.

Optimized construction of low-redundancy cDNA mini-libraries using low-stringency RT-PCR is described cDNAs are generated using arbitrary consensus-degenerate hybrid oligonucleotide primers and nanogram amounts of Schistosoma mansoni mRNA. A number of conditions such as temperature and salt concentration are combined to create balanced, low-stringency conditions that permit a normalized amplification of a diversified, random set of sequences. On average, 350 different sequences are obtained per mini-library, which represents a significantly higher diversity of messages per library when compared to previously published conditions (ie., 20-40 sequences/ mini-library). The optimized high-throughput approach described here is likely to help in the discovery of expressed genes in any complex organism.

A comparative study of repeated sequences in the SM50 gene of some sea urchins

Spicule matrix proteins of sea urchin embryo are the specific products of the micromere / primary mesenchyme cell (PMC) lineage, and are considered to be involved in spicule formation (Wilt, 1999). One of these proteins, SM50, has been described for three species: Strongylocentrotus purpuratus (SP), Lytechinus pictus (Lp) and Hemicentrotus pulcherrimus (Hp) (for references see Wilt, 1999). The nucleotide and amino acid sequences are well conserved in these species. SM50 proteins of these species have repetitive amino acid sequences in the carboxyl-terminal half of the proteins. Therefore, examination of SM50 sequences, especially the repetitive sequence region, in various species will help an understanding of the process of sea urchin ontogeny and evolution. In this study we tried to amplify, by PCR, the SM50 sequences of species for which no sequence data are reported.Total DNA was extracted from the sperm of sea urchins by standard procedures. The purified DNA was subjected to PCR to amplify the repetitive amino acid region and its upstream region. The primers were designed based on the highly conserved sequences in the reported SM50 as Consensus-Degenerate Hybrid Oligonucleotide Primers (Rose et al., 1997). The amplified products were gel-purified, and sequenced using ABI PRISM 310 Genetic Analyzer using PCR primers. The determined nucleotide sequences were translated into amino acid sequences and compared among species with a phylogenetic tree constructed by the neighbour-joining method. For indirect immunofluorescent staining, embryos were fixed with 70% methanol and reacted with rabbit antiserum against recombinant SM50 protein.

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.