The identification of novel members of gene families by PCR using degenerate primers has been considered more of an art than a science, so much so that the methods books I've come across have been too timid to discuss the considerations that go into the design of this experiment, much less give a protocol for its execution. At the risk of leading my readers on wild goose chases, I'm committing my methods to paper. The following is based on my reading of the recent literature (e.g. Buck and Axel, Cell 65: 175-187, 1991; Riddle et al., Cell 75: 1401-1416, 1993; Krauss et al., Cell 75:1431-1444, 1993), discussions with several other successful practitioners of the art, and my own experience isolating vertebrate homologs of the
C. elegans egl-10 gene.
Primer designThis is the most important factor in the success of the experiment, and deserves careful deliberation. I suggest diagramming out an alignment of the existing members of your gene family, highlighting conserved residues, and labeling each important position in the alignment with the number of codons that encode the amino acid(s) at that position. An example based on the original members of the
egl-10 gene family is included below, and cited in the following discussion.
Primer degeneracyIn the early days of degenerate PCR some novel genes were successfully amplified using primer pools that were over 1000-fold degenerate. However, primers of such high degeneracy appear not to have been generally successful (with some exceptions, e.g. Giovane et al., Genes Dev. 8: 1502-1513, 1994), and most recent successes have come using primer pools of 100-fold degeneracy or less. Five methods for reducing the degeneracy of the primers are discussed below:
1) judicious selection of the primer sites
The positioning of the primers is a compromise between placing them at the codons for the most conserved amino acids, and placing them at the codons for less conserved amino acids whose degeneracy may be lower. Consider the case of the 3' primers ("3T and 3A") in the example shown below for the egl-10 gene family. At first it might seem more sensible to place these primers 3 codons to the right, where there is a block of 5 out of 6 absolutely conserved amino acids: SY(P/Q)RFL. Unfortunately, this block of amino acids is encoded by 5184 different DNA sequences. The actual primers used were placed 3 codons to the left. At this position only 3 out of 6 amino acids are absolutely conserved: M(E/K)(K/N)(D/N)SY. However, this block of amino acids is encoded by only 768 different DNA sequences.
2) the use of inosine as a "neutral" base
Inosine is a purine (which occurs naturally in tRNAs) that can form base pairs with cytidine, thymidine, and adenosine (although the inosine:adenosine pairing presumably doesn't fit quite correctly in double stranded DNA, so there may be an energetic penalty to pay when the helix bulges out at this purine:purine pairing). Recently, most people have been using inosine in their primers at positions where any of the four bases might be required. Each use of inosine thus reduces the degeneracy of the primer pool 4-fold. However, you risk the occurrence of I:G mismatches, and therefore must assume that exact base pairing at other positions in the primer will overcome such a problem. Most oligo synthesis facilities will make inosine-containing oligos, no problem. I had excellent luck with inosine-containing primers with the egl-10 gene family, except in the case of the primer "5out", a 20mer containing 5 inosines, (including 2 near the 3' end of the primer) which failed to amplify products even from a cloned egl-10 cDNA. So, perhaps 5 out of 20 inosines is too many.
Using inosine in the primers requires that the DNA polymerase used in the PCR reaction be capable of synthesizing DNA over an inosine-containing template. Taq polymerase is capable of doing this, but some others (e.g. Vent) appear not to be able to.
3) using multiple separate oligo pools at a single position
In an effort to use primer pools with the lowest possible degeneracy, it is sometimes useful to synthesize primers over a particular stretch of codons as two or more separate pools, each of which will have lower degeneracy than you would get by synthesizing a single pool including all of the same codons. The pools are then used separately to carry out PCR reactions. For example, the primer pools "3T" and "3A" in the egl-10 example below are identical, except at their serine codons. Sadly, serine is encoded by 6 different codons, TC(A/G/C/T) and AG(T/C). Synthesizing a single pool covering all these possibilities might require a high degeneracy and would necessarily include some non-serine codons. By splitting into two pools (one nondegenerate containing an inosine, the other 2-fold degenerate) I was able to keep the degeneracy low, and avoid all non-serine codons. Another example is shown in the case of primers "5inE" and "5inR", which again are identical except at one codon.
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