My Outer forward, Outer Reverse bp size is 326bp, which shows in my samples. There are two possible mutations: One showing band size of 202 and the other at 175. All of my samples show either no mutation or mutation at only 202. I am playing with annealing temperatures but it becomes difficult when I don’t have any validated sample (Positive control).
I agree that having no positive control is a problem and making a very long olgo as a control is expensive so:
Are you the first in your lab to set up this assay...if not then a previous researcher may have found a positive sample. Is this a clinical assay so that your, or another, hospital may have some positive sample that they can share.
or is this a research assay that has been set up elsewhere so you can ask the authors for a small dna sample to help your setup.
Is this 175 mutation a common mutation in your population in which case you could amplify the dna of 10 patients and sequence the amplimers to find a positive sample
Those are great suggestions from Paul. Without positive control it is complicated. I have a technical recommendation for the gel: I think the shorter bands are quite weak and also will run close together in your setup (E.g. sample 1 and 4, there could be a very weak 2nd band below the 200 bp one, it is not clear but possible), so i would recommend using a higher percentage agarose gel (2.5%, maybe 3%) and maybe a longer gel, so that you don't loose signal at the bottom during the run and can see the bands better. And if you have another molecular marker with more bands in your desired range, maybe even some other pcr frament of a similar size than 175 it can help you spot a weak band of interest better according to size. I m not an expert in this technique, but I think as the 326 bp fragment is much stronger than the 202 bp one, you could go down with the outer oligo concentration in general vs the inner ones. This you can optimize with the 202 fragment. I m not sure, but maybe if the 175 bp pcr also works alone, you could start the optimization using just a simple pcr with the inner and outer oligo corresponding to the 175 bp fragment. If that works, it will also be a size control and allow you to id a positive sample as control. good luck!
I agree with what Daniela says and in addition do not run the samples so far in the gel. You are not interested in the high molecular weights so running half as long will still separate the small band and the intensity of the small bands will be stronger, An increase in the inner primer concentration might also result in stromger specific bands
Thank you Paul Rutland and Daniela Liebsch for your worthy suggestions. Just like to add I am the first in my institute to do and I am doing this from our hospital's patients DNA for identifying the SNPs in them for the first time. Plus, we have no strong database regarding this SNP in our country so I can't say the 175bp mutation that i am looking for is common or not. But I am hoping to find at least one out of many samples I have been experimenting with. Also, the primers are not designed by me we are using them from a study that designed those in-silico and are not validated by experiments. There is a huge difference between the melting temperatures of each primer in one SNP set and I think this may be the reason why I can't get all primers to work on a same annealing temperature to show mutations. I am currently considering going for a stepdown/touchdown PCR but don't know if it is worth considering. Lastly, I followed Ye et.al's suggestion for primers ratio as well and it showed me more prominent bands but again I could only get mutation at 202. I will run it on 2 to 3% agarose on a long run and use a fragment for 175bp to save time and resources as you suggested. Please let me know if a touchdown PCR would work, and if yes, I need some guidance to set it up. Many thanks! I am attaching the melting temperatures, GC content, and band sizes for my primers for more detail:
I agree with your suggestions @Daniela Liebsch and i learned alot from. But i just need clarification about something because i am still starting experiments involving PCR. You mean pimers by oligos . Rigjt . And And in this case . How he can get Oligos or primers that amplify only the 175 bp mutant without having the mutant in the first place? I am just improving my ideas so please give me your ideas according to your experience. Thanks in advance
Hi you can try try dbsnp at https://www.ncbi.nlm.nih.gov/snp/ for the snp information, Also I see that you have inked out part of your primers. When you calculate the Ta you must use only the part of the primer that can anneal to the template dna so if you have added cloning sequences ignore these for annealing information
Mishal Shaheen the primers you are using are the entire sequence, right, 28 bp roughly that would make sense for the Tm you write. I don't think the temperatures are so different, i can't see that as a reason. The outer primers match 100% but the inner ones have 1 mismatch, so the temperature for the initial annealing steps will also go down a bit and be similar to the outer ones. And IF and IR are also similar, and one of them works. I am a bit skeptic about touchdown, because you get a pretty specific fragment already, and actually you can use a higher annealing T later (because after a few initial rounds of amplification there will be more template that already has the altered primer incorporated, so you could go up a couple of degrees.) What temperature do you use? 65, 67? I think oligo concentration is a better shot for optimization. If you want to try touchdown, here is a reference for how to set it up: Article Touchdown PCR for increased specificity and sensitivity in P...
As mentioned I would see what temperature works well for the short fragment alone only using outer and inner primer for that fragment (but check the annotation, i think the actual 175 bp fragment is with IF and OF not as mentioned in the table. If i m not wrong the 175bp mutation corresponds to the reference human genome (i don't know anything about humans but maybe with that in mind you can get a control from some colleages somewhere, a clone or so?). If this strategy doesn't work, i think your polymorphism is also inside an ApaI site (GGGCCC), if this is true, you can use the outer primer fragment and digest with ApaI to see if you have the polymorphism or not.
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