CODEHOP:
COnsensus-DEgenerate Hybrid Oligonucleotide Primers

Getting started

Blocks from protein sequences for CODEHOP input

Start with a set of related sequences or a multiple alignment in Blocks, Clustal or FASTA-alignment format. For a set of related sequences, get a ClustalW alignment using either the EBI Clustal or the BCM Search Launcher multiple sequence alignment site for global multiple alignments from which blocks are made, or use Blockmaker to make blocks directly. Clustal-generated alignments are copied and pasted into the Multiple alignment processor window in Clustal (include the word 'CLUSTAL' from the heading) or FASTA-alignment format, and choose 'Submit the sequences'. The processor carves out blocks from fully-ungapped regions that are at least 10 residues wide and puts them into Blocks format. Using Blockmaker (Motif or Gibbs sampling) provides blocks that are ready to go for CODEHOP design. Either way, there will be several options that you may find worthwhile, such as viewing logos and trees to aid in the evaluation of the resulting blocks. If you like the blocks, then by choosing the CODEHOP link, your blocks are inserted into the primer design window.

Designing primers

Click on "Look for primers". This is the default report. There are then ways of reducing the stringency if you don't get predictions, or if you don't like what you get. We think that you should first raise the degeneracy to 256 or higher (if you dare) and retry. Next, you might raise the strictness of the core region, for example to 0.1 or 0.25. You might have a favored sequence(s), in which case, rather than raising the degeneracy or strictness, raise the weight of your favored sequences (to bias the primer) by working in the Web box on the last column (max weight is 100; so you might upweight your favorite sequence to, say, 200 or 400). You can also remove individual sequences from the Web box by down-weighing them to 0 (in the last column) if they are too divergent or misaligned and so prevent finding a solution.

Clamp extension

Using the CODEHOP strategy, primers cannot extend beyond the limits of the blocks. Therefore, you may see the message "CLAMP NEEDS EXTENSION" if the resulting melting temperature is too low. In this case, you can copy and paste the primer into this oligo temperature calculation site. Add residues to the 5' end until the desired temperature is reached. Possible ways to select residues to add include: Select them arbitrarily; select codons from one of the sequences represented in the blocks in the regions flanking the blocks ( reverse translation site); re-make the blocks leaving out some sequences or using a different multiple alignment method to get an extended alignment.

Cycling conditions

For amplification, we recommend using AmpliTaq Gold with a 9' preheat (this provides an automatic hotstart - a hotstart of some kind is important). We have had success using the time-release feature with addition of 15-20 extra cycles. The CODEHOP strategy of Rose, et al. (NAR 26_1628-1635) is different from the usual degenerate PCR, and it is desirable to keep annealing temperatures high - even 60^oC may be OK if you have a >60^oC clamp. We recommend trying the highest temperature that yields a clean PCR product. We have used "touchdown" PCR down to Tm-3^oC or lower, say from 63^oC down to a good clamp annealing temperature in -0.5 -> -1^oC increments, and the remaining cycles are carried out at the 53-57^oC clamp annealing temperature for a 60^oC clamp. The intent of the touchdown is to give the correct product a head start, because it is likely to anneal at a higher temperature than any failure product. Once the clamp 'takes over', then all primed products, whether correct or not, will be on an even footing, so we try to keep the stringency high in all cycles. With luck, it should not be necessary to gel-purify product, but rather you may clone directly from the reaction mix if a single band of the expected size is obtained.

Genes identified using CODEHOP.

Please let us know if you have any useful tips to pass on based on your experience using CODEHOP-predicted primers.

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