Which electrophoresis methods can be used for the analysis of SSR polymorphism for the assessment of genetic diversity? Is 3% agarose gel electrophoresis is reliable?
I've never tested this in a gel format. But, in microchips, we have had good results with hydroxyethylcellulose (HEC) and polyvinylpyrrolidone (PVP) mixtures. Both can be conveniently bought as powders from Sigma or else and mixed in the ratio that gives the best results for your application.
Can you please mention which crops or species you are going to asses the genetic diversity?
Well, Please consider the expected size of DNA fragment before making decision about agarose gel percentages. And, if it is rice, 3% agarose gel with 60~65 mins run is enough to get polymorphic alleles. Thanks
The link below to an article maybe interest you: "Robust and inexpensive SSR markers analyses using LI-COR DNA analyzer (2013)".
[ http://www.ncbi.nlm.nih.gov/pubmed/23546793 ]
In its abstract:
"The LI-COR platform offers both qualitative and quantitative improvements over the conventional assays based on agarose and polyacrylamide (PAGE) gels with DNA stained with ethidium bromide and silver or radiolabeled."
if you are going for preliminary studies and are not able to detect the polymorphism (if for few base pair changes), then you can go for metaphor gel.
Both Polyacrylamide and metaphore gel can provide better resolution then agarose gel.
You can use agarose 3% gel running in TAE 1X or TBE 1X buffer (TAE for large fragments and TBE for smaller)
Also depends on ploidy and heterozygosity of the material you are testing and size of alleles per marker you are expecting. If you give us this information, I think we can help you more. Someone else asked you about crop or species.
I agree with Jorge Nuñez, the species and ploidy are determinants. In our lab the SSRs are separated by electrophoresis using 3% Metaphor® agarose for 2 h at 70 V with 1X TBE. However you need to take in account several things. We work with stone fruits, mostly 2n, and generally we perform previous SSRs screenings to determine the allele sizes, since is really difficult the lecture of profiles lower than 15 bp of separation. Finally we choose that SSRs with separation bigger of 25 bp, avoiding errors in reading. Metaphor is a suitable method if you do not have the opportunity of use sequencers (LiCor, ABI, etc.) Good luck.
Microchip applications should be an alternative, as Cedric Hurth suggested. MultiNA by Shimadzu would be one of the platforms you can look at. You do have to use a specific buffer for certain size ranges. You can also try capillary platforms that are dye based, instead of fluorophore based like Fragment Analyzers by Advanced Analytical. Good luck.
I agree with the msr Krishna answer 3% of Agarose and metaphor give you high resolution. but metaphor give you high resolution than Agarose .
Thank you all for your response. I have tried both 3% agarose and metaphor. Both give separation in SSR analysis. Metaphor is better in case of resolution.
I have used 4% metaphor agarose gels and the electrophoresis was runned by 4 hours. The results showed an optimal separation of fragments, so, was easy the allele identification.
if you have sequencer facility/or manage for it, you can do multiplex,no gel analysis is needed.
3% agarose and metaphor both give separation in SSR analysis. Metaphor is better in case of resolution.
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