I am trying to amplify seven SSR loci, that I used to genotype my study species individuals, in 2 out-group species (within same genus and within same family). I am getting amplification for 2 loci in individuals belonging to the same family using the same protocol that I optimize before but other 5 loci are not amplifying. Also, no loci amplified in individuals belonging to the same genus. DNA concentration for out-group individuals within the same genus = 5ng/ul and within same family = 3 ng/ul. I would appreciate it if anyone could suggest anything about this problem.
Note: I have already tried touchdown PCR annealing temperature variable
Thanks
SNIP regions or repeat might appear inside variable zones of gene of new individual if primer arrangement was not tested with enough cases before. Maybe primers design-arrangement should be checked again if your controls amplify normally.
If you have access to a temperature gradient PCR, try optimizing the temperature and MgCl2 concentrations (try a range of concentrations from 0.5 to 5.0 mM for each temperature). This often helps
I tried to vary MgCl2 amount yesterday. This image shows the result. I did not use negative control (so I am not sure right now whether well number 3, 4 and 6 is showing me real band or primer dimer). MOST PROBABLY those 3 wells have real bands in my onion. I do not have expected size because these primers are designed from HiSeq sequences. What do you guys think? Thanks much for suggestions :))
Dear Niraj
Cross-amplification amongst the species is always an issue with respect to SSR markers and so you can design and try multiple primers (priming regions of the same flanking regions) for a single marker as you are looking for only 5 markers. Also suggest you to increase your template DNA concentration along with the denaturation time.
Good Luck
If in doubt, you could always sequence those PCR products to see if they contain the expected SSR..This might also help desing a primer with more specific 3 prime ends
It is possible that you still need to do some adjustment of the PCR conditions for samples that do not amplify. You should try to move/change each componenet of your PCR. You should try to change e.g. amount of template DNA, number of cycles, amount of polymerase, try also different polymerases and so on. If you will not notice any improvement, then it is probable that you have „null alleles“ in those species for those loci. It is possible that there is a mutation on the primer-site region (one or more nucleotides are changed and the primers are not perfectly complementary to DNA strands). Than the PCR is not successful. If this is your case, you will not get the products from those loci. Or, you can try to design new primers with several degenerate nucleotides at different position.
You could try to lower the anneal temperature to something very low (e.g., 40 or less). That is an approach I often use to see if I can get any amplicon when I consistently get no amplicons.
Hello Niraj,
Padmakar is right. Transferability of SSR markers between species is not always good. As far as I know it may work within a plant genus (or maybe even within a family), but for fungi SSRs are more species specific. Without knowing your target organism it's difficult to answer more specifically. You might want to look up some info in the literture about SSR compatibility in the species you are working with. It may turn out that new markers will be needed rather than investing effort in PCR optimisation.
The comments from both Anders and Zoltan are spot on. I would try Anders suggestion but I would not be too surprised if amplification was still non-existent in the more distantly related species. You mentioned HiSeq sequences and primer design. You should still have an idea of expected size (given you've designed primers that flank the SSR) but were the sequences only for a primary target species or do you have consistently sequenced regions among some of your taxa? If the latter, how do they align and how conserved are your primers? This might help you should primer design need to be reconsidered for additional taxa.
Clearly you have annealing issues. I would guess that there is a mutation in the flanking region where the primers should anneal. Can you design species-specific primers?
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