Hi,
I am studying the antibiotic resistance and I am amplifying a class of integrase genes that are known to be associated to multi-drug resistant phenotypes.I have done PCR with primers for this integrase gene and I tested different bacterial isolates for the presence of the gene. I attached the picture of the gel I have. Each well corresponds to the PCR product of each bacterial isolate. I don't know how to interpret the results of the second, seventh, eighth, ninth and eleventh well. Indeed,I have a band at the same position as that in all the other wells but it is much slighter. Furthermore I have different slight bands corresponding to different size products. I thought they can be aspecific product but the annealing temperature is quite stringent (and if it were the result of aspecific product, maybe I should see these bands also in the wells where the bands are clear?). I was suggested that it can also be due to the fact I am amplifying an integron, so different genes can be integrated leading to different product sizes. Does it make sense for you? Anyway, why do I obtain a much slighter band?
I do not know really but a possibility is that these samples have a polymorphism under the 3' end of one primer. Then the specific pcr works badly and there is plenty of primer trying to find something to do so maybe we are seeing something like a RAPD effect . You will notice that there is also a primer dimer only on the odd samples. This is testable maybe by running the same pcr with only one primer in each tube on one of these samples and see if you get the same band pattern. You do often fing that when a pcr works well for one amplimer the other bands disappear eg when primer dimer amplifies then often there is no visible proper product
Thank you Paul for your answer. Can I ask more in detail what does "polymorphism under the 3' end of one primer"mean exactly?
Thank you again
a polymorphism is when there can be more than one base present in a sequence at the same site. So one species or some of the population perhaps have an A at a position and others have a G. Then if your primer was designed against the sequence with an A ( so the primer has a T at that position) then the primer will not anneal to the other populations dna which has the primer's T trying to anneal to the sequences G. If this happens near the 3' end of the primer where extension starts from then often there can be no pcr extension as the end of the primer is waving around and not stuck to the target dna. Then you get no ( or poor) pcr product but the primer may anneal elsewhere and produce the non specific bands. There are millions of polymorphisms in the human genome ...they are changes often in introns or inter genic areas and usually have no effect on anything unlike mutations which are really just polymorphisms which do have an effect on the lifeform
simply change the DNA enzyme to see how it changes. Try Tag low-fidelity enzyme first.
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