I want to standardize detection of mutational changes in particular Genes by SSCP. I am using nested PCR for that but facing problems of weird amplification pattern in NTC reaction as well. I tried best to avoid any type of contamination and cross contamination as well.
Hi Adil Husain
you must list every technical steps, things and buffers used. a particular attention of the rooms where you manipulate with not obvious step. why using nested PCR? is it the reason for contaminations?
fred
Dear Adil, it difficult to give you a real good solution because nested PCR always leads to high probability of contamination. I have experience in this but it was difficult to receive clear NTC. I know that it is more expencive, but you can try NGS to detect changes.
Is your weird pattern a smear of high molecular weight fluorescence on the gel?
Contamination is very likely as every 10 cycles is 1000 times more product so a nested pcr of 30cycles plus another 20 cycles is 10exp15 times more product. This is a lot of product and the tiniest amount loose around the lab is a huge number of molecules and ideal for further amplification.
The smear comes when the product not the primer is in excess and product re anneals at various points with other product molecules and a longer product is made.
Contamination clean up is hard and time consuming but if your experiments allow it moving one primer just outside of the current amplimer means that it cannot anneal to the product and a clean new, larger product is the result. If size is a problem( and it often is in sscp) then just move both primers in one direction 20 or more bases and you should get a clean product and negative NTC sample again
Dear Paul,
In above problem discussed, NTC gives same band size product as DNA samples do.
if you had a clean ntc when you first started using these primers then you do have contamination, you can throw away all of your reagents and order more. clean up your pipettes and working area and hope it solves the problem or make new primers both sets having onw primer outside of the first round original primer set because at the moment you do not know if the contamination is first or second round product, If time or money is critical then you could try moving to another workers area.use their pipettes and all their pcr ragents and a new unused aliquot of primer and hope that your primers are not the contaminated reagent. Contamination will always be a problem with pcr and especially nested pcr so if you can get a good product with just first round pcr it would help . It also helps never to have pcr setup and post pcr work in different areas so set up in one area with one set of pipettes and open the tubes and run electrophoresis in another area and never bring the pipettes/tubes back from the gel running area into your setup area. My guess is that opening a pcr tube sprayed microscopic droplets of pcr product into your working area or that you used the same pipetes to set up gel loading and your pipettes are contaminated and will need stripping down and cleaning
Thanks for your answer @Dear Paul. We are using PCR hood as working area and dedicated pipettes for Nested PCR. We had changed both primers sets(designed by dCAPS) & reagent stocks as well but facing same issues. Is PCR kinetics cause these type of unwanted amplifications.??
If you have the opportunity to try this type of area, test your PCR.
dust or something in the air maybe...
fred
Do you mean dcaps Adil.It is a mutation method but not sscp as in your original question ?
Anyway no I do not think that pcr kinetics is to blame. If you are using good pcr practice for the nested pcr then it just suggests that the contamination has occurred in the first round of pcr amplification and from that point onwards all amplimers internal to the contaminating product will be amplified from that contamination. I think that even dcaps primers which are deliberately mismatched to the target sequence will amplify from the contamination because you have deliberately lowered the annealing temperature for the dcaps reaction to ensure that the primer does anneal. If the pcr started clean but then started to produce product in the ntc it is definitely contamination. There have been cases whenpeole make their own taq and are amplifying genes that exist in the TAQ bacterium that they can produce an amplimer from the taq dna but I am thinking this is not the situation in your case
ok that make some of what I suggested wrong as you cannot move all primers.
I think a major clean up and replacement of regents is still needed but it might still be useful to know which amplimer is causing the contamination. If it is the first round amplimer then clean up is the only option ( or move to another lab and do everything there with new reagents) but if the contaminant is dcaps amplimer then you could move the common (non mutated) primer outside of the current dcaps amplimer and this will work as contamination will not amplify. It just makes the difference between cut and uncut slightly more difficult to resolve in agarose gels.. If the amplimer contamination is first round product then it is clean up time.. If you have guaranteed clean primer stocks you can move to a colleagues working area,use their pipettes and pcr reagents and pcr machine and things might work ( plenty of NTC tubes) while the clean up is in progress. In a previous answer to a contamination problem a colleague reported that it cost his company $60,000 to clean up pcr contamination in his laboratory so hopefully you are research orientated when time is not costed in the same way. good luck
Paul
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