I did PCR for fusion gene by using cDNA and sent the amplified product directly for sequencing in both forward and reverse direction. When I blast the sequencing result on NCBI, 90% of my results didn't mach to the gene of interest. My mentor thought that it would be difficult to analyze fusion gene by using software and databases so he advised me to match all the sequence manually with the reference sequence, I did so. I found my primer sequence in my sequencing results along with small chunks of similar sequence. Chromatogram is not good enough. Should I consider my sequencing results positive for my fusion gene or not?
Hi, Thank you for the response.
I did PCR for two different exons of EML4 (as a forward primers) and use ALK as Reverse primer for both EML4 primers. For both exons, I used the normal Reverse-Transcriptase-PCR conditions using normal Taq polymerase (Sigma), However, I used Betaine for amplification of one primer pair. Product length is 1100 and 650bp for the two primers. My PCR product length is exactly what was predicted and amplification was very neat and bands were quite sharp without any secondary structure or dimers. Chromatogram for first half is quite better but overall you can't consider it very good.
1. If your primers annealed to non-specific region and amplified a band, when you sequence this band, you will see the primers present, but the other part of the fragment is not your gene-of-interest.
2. You could also picked up a similar allele with some conserved region, not the exact gene of your interest
3. Do a sequence again, sequence in both direction and assemble the results to obtain good reads; then you compare again to your gene-of-interest
Primers are specific according to NCBI database. Apparently, PCR is giving the exact size amplification and without any secondary structures. I did PCR 3 times for all positive samples, improve the amplification but still sequencing results are ambiguous. May be because amplification belongs to 2 different genes fusing together. Few of my samples showing 80% similarity with gene of interest on NCBI, also showing primer in sequencing results when i checked it manually.
If you sequence your PCR fragment from forward direction you may find the reverse primer sequence at the end and vice-versa. If you sequence from forward and find forward primer sequence in your results and the Chromatogram is not good enough, the sequence result could not be considered positive.
yes, I found the reverse primer sequence in the Forward one and vice-versa. For normal gene mutation or gene screening, we can relay on neat peaks of chromatogram but just want to know whether the same rule applies to fusion gene as well or we may have any other way to rule this out?
I thought it depends on the qualities of your PCR template mainly, especially for checking a fuse-gene. If your DNA template contains both tumor cell DNAs and normal cell DNAs, your PCR result might have some mix peaks in the Chromatogram except for the case that only fuse-gene could be amplificated in your PCR reaction. Otherwise, the heterogeneity of tumour cells also might make your Chromatogram peaks not good enough so it is necessary for checking your Chromatogram carefully to find the possible reasons.