I am trying to find gene fusion in cancer cell lines. I am using cDNA in conventional PCR. Forward and reverse primers belong to 2 different genes so I don't know the product size. I found a gene fusion around 500 bp size but the problem is amplification is very poor to confirm it by sequencing. My primer has two Tm values, one around 70 and other one around 58. I don't know which value should be considered for annealing. I tried 52, 52, 57,58 and 63 degree annealing but still the amplification is not good. I am using 10x PCR buffer with MgSo4 so I can't play around with MgSo4. Any suggestion to rule out this problem?
Even if it is already in the buffer you could add more Mg salt. Are you doing some positive control on cDNA? Do you have reagents like betaine?
I don't have positive control for this gene fusion, but to check whether my PCR is working or not, I use a primer pair that amplify well in cancer cell lines. You can see the gel image attached, first from the right side is control primer, while 3rd is my gene fusion.
Since your primers have very different Tms (70°C and 58°C) I would strongly recommend to design new primers with a more similar Tm, e.g. 62+/-3°C.
Conducting a google search for recommendations for primer design will retrieve several useful links to optimize your primer design
This is tm of a same primer. there are 2 tm mention on a primer. My forward primer has Tm 58 and reverse primer has Tm 56. I have tried annealing from 52 to 63 but the amplification is still very poor.
my thermal cyclic conditions are:
94-4
94-30sec
52-63,- 30 sec
72-90 sec
72-10min
Do i need to change these conditions?
Sorry, I misunderstood you. No, the PCR conditions are fine and your bands appear to be specific. How many cycles are you running? Add a few more and/or use more template.
I tried 30 cycles and 35 cycles.
It will work if I add some PCR stabilizer like Betaine?
how do you know it is the fusion gene ? if you know the sequences, you may design a primer to confirm it by qPCR (product is about 80-200bp) , run gel and sequence.
because I have designed the primer for 2 different genes, and both genes are suspected for gene fusion. By conventional PCR, I am just suspecting about gene fusion but i want to confirm by sequencing.
Betaine is generally used for the amplification of GC rich regions; you may try to add it. Another option is to test different Taq polymerases, and you could add a few more cycles.
Is it ok to use more than 35 cycles??
I have tried with increasing MgCl2 and waiting for the result. I will try some other polymerases also.
I don't think its a problem with input or the number of cycles (not even a slight band on your gel after PCR). You may just want to recheck your targets or make multiple primers per gene at some intervals and try some combinations. Gene fusions can differ and interrogating different regions along the gene may give you what you want.
How did you find this 500bp gene fusion?
I have used 3 different primer pairs for EML4 and ALK fusion, as you can see from the gel image that top portion(I used 2 primer pairs with 7 cDNA) has no band, but in the lower image of gel, first primer is giving band with this 2 samples that is around 500bp.
I tried extra MgCl2 today but it doesn't effect the bands but it causes some non.specific amplification. tomorrow i will change the polymerase. I don't know what else i can do to improve it.
Today I increased the extension to 6 minute and used a PCR enhancer. I got amplification, still the bands are not very sharp but better than before. I hope to improve it more. Thank to all for giving me your valuable suggestions.
Can anybody please suggest me any useful conventional PCR kit that can be used in gene fusion identification??
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