To track the Tos17 insertion mutation, I used 3 primers (1 forward and 1 reverse away from the insertion and 1 forward on the insertion site). In wild type I got single band; however, in heterozygous individuals I got >3 bands on the gel. I wonder is it because either of the primers (F&R away from the insertion) didn't bind to the DNA that give rise to one long extra DNA band?
hard to say without a picture but If one of the bands is bigger than it should be then you have a heteroduplex of normal and mutant dna. This has a bubble in its structure that makes the product run slowly in the gel. To test this idea either mix normal and mutant ,heat and re anneal and see if the band appears or cut out the large band from the gel and denature it and see if it reforms as 3 bands not the one that you started with. It sounds like your pcr is working perfectly. The third band is diagnostic of a heterozygote under the pcr conditions that you are using
hard to say without a picture but If one of the bands is bigger than it should be then you have a heteroduplex of normal and mutant dna. This has a bubble in its structure that makes the product run slowly in the gel. To test this idea either mix normal and mutant ,heat and re anneal and see if the band appears or cut out the large band from the gel and denature it and see if it reforms as 3 bands not the one that you started with. It sounds like your pcr is working perfectly. The third band is diagnostic of a heterozygote under the pcr conditions that you are using
as Paul Rutland suggested, a picture with size of amplicons and insert would help us considerably. but considering you have only one variant (the insertion), for WT with 3 primers you should have only one band on gel, and 2 with mutant. little precision on species would also help.
fred
Probably it's due to unspecific annealing try gradient annealing temperature , also can attach the picture and the PCR program .
I agree with Paul. it is possible that mutation co-inherited with a privet SNP in somewhere in the affected gene or out side.
It is reasonable to have three bands in heterozygous state. Having more than three bands would probably be due to unspecific annealing, etc.
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