I want to detect 15 bp of difference in DNA fragments of 350-385 bp length. Which method will be most useful? Agarose, PAGE or primer labeling and genotyping?
Hey guys, just resolved a 14bp difference on a 3% agarose gel prepared and ran in TAE buffer, please see the attached picture.
Hi,
It depends. Agarose is the easiest alternative. A difference of 15 bp is detectable in a 2% or 2.5 % agarose gel. However, you should use positive controls of your bands and you should run the electrophoresis at low voltage and avoiding heating of the gel.
PAGE has better resolution but is more time-consuming.
The easiest way is to use agarose, but not the regular type. There are some agarose that can be melted at 4-5%. You mus cast long gels though. This expensive however. The cheapest way is to use PAGE with silver stain, but this is tricky. Primer labeling is the method of choice if you have the needed equipment.
Hi Pankaj
You can run your samples on a high(er) percentage (2-3%) TAE-agarose gel. To make the gel without bubbles, microwave in a pressurized container (ie, Pyrex bottle with lid fully tightened) and slowly release the lid once the agarose has fully melted. My previous lab used this method (RFLP) to genotype a 4bp deletion in the filaggrin gene.
Joe
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I agree with Sergio R. Matioli.
I think primer labeling is the most reliable method (at least, I have had very nice experiences with it). But I suppose it depends a bit on what your throughput is: will this be for routine samples, one time samples, and how many samples?
If this is a one time thing for a few samples I would try with agarose or PAGE. But if you intend to do this a lot, save yourself the trouble and use labeled primers.
you could use either agarose or PAGE but first make the size a bit better so find a restriction enzyme that cuts leaving you a smaller fragment such that you have a constant band and smaller bands of L and L+15 in a range where 15 bases difference is easier to detect but is still long enough to intercalate ethidium bromide. Somewhere in the region of 100 plu 115 bases or whatever region your agarose separates well in
Run your samples on 2 - 3 % agarose gel, but you use TBA not TAE, because with use TBE your sample will be better visualised. Or use RFLP method, DGGE.
I would like to think it depends how often and how precise the resolution needs to be. In case you will do this analysis as a one time event and you would only need to see that there is a difference in your products, I suggest to prepare an agarose gel to 3-4%. If you analysis must show the 15bp difference precisely, use primer-labeling and run your samples on a sequencer such as an ABI 3031 or whatever you have in your lab.
You wrote:
I want to detect 15 bp of difference in DNA fragments of 350-385 bp length. Which method will be most useful? Agarose, PAGE or primer labeling and genotyping?
If you really do have a mixture of fragments of this range in your sample, then the shortest fragments with the additional 15 bp will not be resolved from the 385 bp fragment. So what is it you have, and what are you actually trying to do?
Thank you all for various suggestions. I tried running the samples on 8% PAGE, but it failed. I did it twice. I have two fragments in the mixture - a 350 bp and a 385 bp fragment.
On the other hand, with a 3% agarose gel, I succeed in resolving the difference.
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