4)
including partial codons at the ends of the primersThe various codons encoding an amino acid or a set of similar amino acids are often identical at their first (and maybe second) positions, but different at their third position. You can take advantage of this by synthesizing only the first or first and second positions of the 3' most codon covered by your primer pools, thus giving you one or two extra positions of exact match base pairs without adding any degeneracy. In the
egl-10 example, the primer pools "3T" and "3A" cover a stretch in which the last codon must encode proline or glutamine. The codons for these two amino acids all start with C, but their last two positions are degenerate. Therefore, only the nondegenerate C was included in the primer pools.
5)
use of codon biasSome organisms have strong biases for using particular codons to encode certain amino acids. In theory you could reduce the degeneracy of a primer pool by only including these most common codons, and taking the risk that the gene(s) you are looking for will follow the organism's general codon bias enough to allow such primer pools to work. I haven't heard of anyone actually using this codon bias strategy in a successful degenerate PCR experiment, but you might try it if you're desperate.
Other considerations in primer design
1) primer length
In the example below the short stretches of sequence similarity among the egl-10 family members forced me to use primers only 19-21 bases long. These are shorter than the primers I have heard of people using in other successful experiments. For example, Linda Buck's primers were 31-33mers.
2) 3' end
People I talked to emphasized the special importance of having an exact match between the primer and template near the 3' end of the primer, although I'm not aware of specific data supporting this idea. For egl-10 I tried to avoid having any inosines near the 3' ends of the primers (except for primer "5out", which in fact failed to give any products), and also anchored the primers when possible with a nondegenerate codon at their 3' ends, so that 100% of the primers in the pool would be able to pair perfectly with the correct template over these last few bases.
3) nested primers
If the sequence similarity in your gene family permits, it is a good idea to make nested sets of PCR primers. That way one round of PCR can be performed using the outside primers, and individual products (or the whole mix) can then be reamplified using the inside primers. Products amplified through both rounds are more likely to be the desired new gene family members, and less likely to be spurious products from sequences that happen to contain a couple of primer annealing sites by chance.
Determining optimal reaction conditions
A number of parameters can be varied to optimize reaction conditions for degenerate PCR. These include: primer concentration, magnesium concentration, template concentration, number of cycles of amplification, and the temperatures and times of each step in the amplification cycle. If each of these parameters is to be independently varied, the number of possibilities quickly reaches mind boggling proportions. My philosophy has been to fix almost all these parameters at the standard levels that have been successful for other people, and to vary only the one parameter that I think is the most crucial: the temperature of the annealing step during amplification.
My standard PCR reactions are as follows:
1.5 µl template DNA (2-300 ng)
5 µl 5 µl 10X PCR buffer (10X buffer=100 mM Tris pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% gelatin)
8 µl dNTP mix (1.25 mM each dNTP)
0.2 µl "ampliTaq" polymerase (5 U/µl)
25 µl dH20
5 µl each primer pool at 20 µM each
total volume 50 µl
In practice, the reactions are set up by placing the primers and template into a 0.5 ml tube, then adding two drops of mineral oil from a blue tip, and adding on top of the oil 38.5 µl of a premix containing all the other components. In this way, it is easy to set up many different primer/template combinations at once. The tubes are then briefly spun in a microfuge to combine the two aqueous phases, and the tubes are immediately placed in the PCR block preheated to 95· for a "hot start".
My amplification program:
95· X 3 min. (hot start)
??· X 1 min. (this annealing temperature is varied to optimize the amplification)
72· X 2 min.
94· X 45 sec.
40 cycles of the above 3 steps
72· X 5 min.
hold at 4·
This takes ~4.5 hours to run on an MJ Research machine.
To test the primers and optimize the conditions, I do a series of amplification runs starting with an annealing temperature of 25·, and increasing in 5· increments until amplification fails to occur. Typically for each primer pair being tested, at each temperature, I run 3 reactions containing different templates: 1) a positive control containing 2 ng of a cloned member of the gene family of interest as template. 2) a negative control containing no template (this is very important-- you don't want to get fooled by contaminants). 3) an experimental reaction containing a complex template such as genomic DNA or total cDNA. For total
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