One of my colleagues questions the results of the SSR analysis I have recently performed. With primer AMS12, we obtained 8 different bands, ranging from 200bp to 600bp. He is insisting that on the gel the bands must be strong intensity to be accepted as good results. Therefore, can anyone explain why SSR DNA markers produce bands that are not the same intensity ?
Hey there,
From my lack of knowledge of SSR, I can say that it is normal that bands of different sizes have different intensity: the DNA dye (EthBr, SYBR...) binds in a way proportional to band length, so the bigger the band, the bigger the intensity assuming you have the same number of molecules for different bands. However, smaller amplicons can be amplified more efficiently than bigger amplicons, so even using the same primer pair, I think it's possible you could get different amplification rates, and then different end-point amount of amplicon. On top of it, microsatellites are by definition repetitive, so I see likely that secondary amplicon structures could affect amplification efficiencies.
That said, my approach (with no knowledge of SSR's) would be to run a control (or a standard curve) of known number of repeats, and if the results are consistent in intensity with your tests, I would say they should be trusted. That control would be then ideal to optimise the PCR so you get the best amplification efficiency, because faint bands could be either negatives or false negatives.
Cheers,
Ruben
i also have no knowledge of SSR but if the GC content of some amplimers is high and some bands are produced by primers annealing at close to their Tm then as small change in pcr efficiency will mean a very large difference in band intensity over 30 cycles
You have to optimise your SSR marker both physical and chemically. Lack of optimization will produce unspecific amplification . Say for eg. you got 8 different bands while SSR Pcr, remember 7 of them were unspecific bands if your marker is monomorphic. If your marker is polymorphic means 6 of your bands were unspecific bands. For each SSR marker there should be a expected product size that can be scored for your analysis or taken to any other downstream process as your wish.
How have your separated the amplicons after PCR- agarose gel electrophoresis or PAGE ?
Dear all,
Those primer i have used are chosen according three papers, who did the same analysis just on their onion varieties. All primers resulted in the expected bands plus that 5 of 7 primers had less or more bands, compared to the literature, which was actually expected. All the analysis were repeated many times, trying gradient and touch down PCR in order to obtain the highest number of bands. After the PCR was optimized,we repeated several times the same PCR to see if we get the same bands and in 90% of the cases we get the same number. However, no positive control was used and the intensity of bands was different (in one primer for example, band 1 to band 8 differ in intensity , where band 3 was double weaker then band 5 and etc...but they were all visible and used for genetic distance analysis with Darwin software. I obtained very good results, just I am concerned about not having positive control and the band intensity.
Mr. Binoy, I can exclude unspecific amplification because of the DNA ladder i used, all the bands were in the expected range (please correct me if I am wrong)?
Mr. Ruben, thanks for the advise, since some of the primers have close annealing temperatures (according the literature), I assume that i can run them both in the same PCR and it can be a positive control. Actually I did it and obtained the expected results, but didn't think of it as a positive control..:)
Dear Mr.Paul, thanks again for the advice, i did most of the time touchdown PCR, in the Tm range proposed by primer manufacturer and literature. However, even the same primers (same sequence) it turned out thatin the literature the Tm was different, four scientific paper had for the same primer 4 different annealing temperature, so i decided to go with touchdown PCR. Few times many the TD PCR gave slight different results. I usually did not do more than 30 cycles in total to exactly avoid primer dimers).
Mr.Rejesh, I used different agarose gel concentrations, but since the expected band size was between 200 and 800bp we decided to used gels of 1-2%, no polyacrylamide gel was used.
Thanks you all for the answers. ;)
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