I'm using PCR followed by restriction digest with xcm1 as a method to screen for a SNP. In WT DNA I get a result which is digested into 3 distinct products, in DNA containing the SNP this becomes 4 products as the SNP introduces another restriction site. However it doesn't seem to digest very well at this site, at least not as well as the others as I get a very faint band and I'm worried I might be missing positive results. Can anyone tell me why this might be happening and what I can do about it?
If you are amplifying DNA from a diploid genome, and the SNP is in heterozygosis, it is very likely that you end up with an important proportion of heteroduplex molecules after PCR amplification, especially if the PCR program includes many cycles after reaching plateau. In every cycle after plateau, there is not actual amplification but mostly denaturing-renaturing of the DNA molecules. Many of them will renature as heteroduplex (theoretically around %50 of them), which will not be digested by the restriction enzyme. So, if the variant is in heterozygosis, you will have around 25% of molecules digested (since WT-WT homoduplex and WT-Variant heteroduplex will not be digested). If this is the case, either try to reduce the number of cycles in your PCR program to the bare minimum to obtain an observable product after digestion, or just cope with the fact that the SNP in heterozygosis will not render a 50-50 digestion.
Hope this helps.
If you are amplifying DNA from a diploid genome, and the SNP is in heterozygosis, it is very likely that you end up with an important proportion of heteroduplex molecules after PCR amplification, especially if the PCR program includes many cycles after reaching plateau. In every cycle after plateau, there is not actual amplification but mostly denaturing-renaturing of the DNA molecules. Many of them will renature as heteroduplex (theoretically around %50 of them), which will not be digested by the restriction enzyme. So, if the variant is in heterozygosis, you will have around 25% of molecules digested (since WT-WT homoduplex and WT-Variant heteroduplex will not be digested). If this is the case, either try to reduce the number of cycles in your PCR program to the bare minimum to obtain an observable product after digestion, or just cope with the fact that the SNP in heterozygosis will not render a 50-50 digestion.
Hope this helps.
I think that’s an excellent explanation by Sergio. Alternatively to test it, you can design to different oligos at the level of the SNP with the two possible nucleotides at the 3' and perform separate qPCR. That will give you equal proportion if you have heterozygosis.
I agree fully with Sergio above as well! His suggestion should definitely be implicated. Another thing that you might consider is the type of gel your are using as well as buffer and conditions under which you are running it. If this addition fragment produced is much smaller for example you tend to see a much fainter band relative to the others due to fragment migration
The band I'm looking for is 500bp where as there are lower size bands which come up with higher intensity, this is what worried me about it in the first place. Thanks Sergio you answer is useful. I have considered re-designing using taqman system but want to avoid it if I can.
In my experience, RFLP analysis of heterozygous samples with whatever enzyme usually yields 50-50 digestion as long as you keep your total number of PCR cycles under 30 (if you do not get enough product with 30 cycles when working on genomic DNA, do optimize your PCR before going any further!). The problem that you encounter is in fact that XcmI is usually giving incomplete digestion of some sites, a problem known since the earlies 1990s when XcmI digestion was used to generate T-vectors for PCR product cloning (e.g. Nucleic Acids Res. 1991; 19:4560). I believe that incomplete digestion happens because XcmI has a degenerate site (5'-CCANNNNN_NNNNTGG-3', with'_' indicating the position of the cut) and the cleavage efficiency of different sequences around the cleavage point varies. I suggest trying another enzyme (if at all possible) or resorting to other techniques. Good luck!
you can use CEL I or Surveyor Nuclease that will cleave at mismatches in heteroduplexes, and that can be resolved on a gel:
http://www.biotechniques.com/multimedia/archive/00036/BTN_A_04364PF01_O_36417a.pdf
I fully concur with Francisco del Castillo.Try another enzyme or another technique. Go for ARMS / MS PCR
Dear David you can apply a latest method of PCR called CTPP method which is more robust than any other protocol today that are available like restriction digestion, arms PCR,etc . if u wnat help on that you can contact me at [email protected]
Dear David you can apply a latest method of PCR called CTPP method which is more robust than any other protocol today that are available like restriction digestion, arms PCR,etc . if u wnat help on that you can contact me at [email protected]
Which band is faint? Is it a digestion product band? If so are the the two bands of produced by the SNP of the same intensity? If so you may have a heterogenous mixture of WT and mutant species. If this should not be so, do a time digest to optimize the digestion; although I think that you your DNA is heterogeneous.
I agree with Sergio' and Francisco' explanations and suggest you to test another method , specially if you want to screen many samples for your SNP : Taqman assay, Surveyor assay, hybridization probes or Simple probe assay are easy, convenient and efficient methods.
Good luck !
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