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

Abstract

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).

Figure 1

Block representation of a highly conserved region within the family of DNA methyltransferases. The ungapped multiple sequence alignment of Block D of the DNA methyltransferases was obtained from a comparison of eight DNA methyltransferase sequences: MTCH_ARATH, AAC02665 (CMT1); MTCH_CARAR, AAB95486 (CMT1); MTDM_ARATH AAF14882 (MET2); MTDM_MOUSE AAC40061 (MET1); MTDM_HUMAN AAF23609 (MET1); MTDM_XENLA JC5145 (MET1); MTDM_CHICK Q92072 (MET1); MTDM_PARLI CAD43079 (MET1) using BlockMaker. Information regarding the size of the block and its position within the original sequences is indicated. In addition, a relative sequence weight has been assigned to each aligned sequence to reduce redundancy and emphasize diversity in the multiple sequence alignment (5). As shown, the sequence weights for chromomethylase sequences, MTCH_CARAR and MTCH_ARATH, were increased four times to bias CODEHOP design towards chromomethylases. The block output of BlockMaker is hypertext linked to the CODEHOP designer.

Figure 2

Anatomy of a CODEHOP PCR primer. A CODEHOP PCR primer is targeted to an amino acid sequence motif within a block of amino acids conserved between different members of a protein family. In this example, the sequence logo representation (9) shows the amino acid conservation within the DNA methyltransferase Block D (Fig. 1), which was successfully used to isolate Arabidopsis thaliana CMT2 and CMT3 proteins (1). The height of each amino acid is proportional to its degree of conservation. The CODEHOP PCR primer is targeted to the highly conserved ‘PCQG’ motif and consists of a pool of 16 related primers in which the 3′ degenerate core contains all of the possible nucleotide sequences encoding the ‘PCQG’ motif. The 5′ consensus clamp, immediately upstream, contains the most probable nucleotide at each position flanking the ‘PCQG’ motif.

Figure 3

The CODEHOP PCR strategy. CODEHOP PCR primers target conserved motifs of 3–4 amino acids using a short 3′ degenerate core containing all codon possibilities for the motif. Annealing of the degenerate core of the primer to the starting template is stabilized by the 5′ consensus clamp. During subsequent rounds of amplification, annealing of primer to PCR product is driven by the identical match with the 5′ consensus clamp between the incorporated primer and primers remaining in the pool. This strategy permits amplification of distantly related sequences with limited homology to known family members.

Figure 4

CODEHOP designer output. The biased Block D of the DNA methyltransferases (Fig. 1) was used to design CODEHOP PCR primers using the default 60°C (A) or 65°C (B) annealing temperature parameter. The consensus amino acid residues and predicted CODEHOP PCR primers (5′–3′) are shown. The preferred CODEHOP has 16-fold degeneracy. The change in the 5′ clamp length obtained with the higher annealing temperature setting in (B) is evident. The anti-sense CODEHOP PCR primer designed from the complementary strand of the DNA encoding Block F of the DNA methyltransferase sequences is shown (C). The primers shown here are very similar but not identical to those utilized in our previous study to identify new DNA methyltransferases (1), since the codon usage table for A.thaliana has changed since 1998. The degeneracy and length for each core and the length and annealing temperature of each clamp are indicated.