Hi guys,
I'm currently doing a research in BCR-ABL. I'm trying construct the Fusion transcripts that causes philadelphia chromosome. For example, presence of fusion transcript e1a2 leads to ALL. In order to create the fusion transcript, I designed primers to amplify the exonic regions of BCR and ABL individually and once amplified, to fuse the amplicons using BCR/Forward and ABL/Reverse primer.
Unfortunately, the individual gene amplifications hasn't worked and it just gives smeared appearance for a specific temperature. The template for the PCR is cDNA synthesized using MMLV.
Appreciate if anyone could help me with troubleshooting this issue.
The PCR cycle is,
Initial denaturation 95c/5min/1cycle
Denaturation 95c/30secs
Annealing 60c/64c/67c/ 30secs
Extension 72c/1minute
Final extension 72c/5minutes/1cycle
Hold 4c/infinity
Denaturation, annealing and extention went for 35 cycles in veriflex.
The gel image is attached for reference.
Hi Harsha,
One trick I was using when doing fusion PCR was to perform 5 cycles of amplification without primers, then add primers to resume the PCR protocol for the remaining 30 cycles; this allowed the fragments to anneal in their overlapping 3' regions and the polymerase to complete the fusion protein without interference by primers.
Hoping that helps, best of luck!
Jerome
How big is your expected product? The general rule is 1 min extension for every 1000 bp of DNA. You also might want to try a gradient of annealing temperatures to see what works best.
Good luck!
Hi Harsha. Great suggestions by Jérôme Teulière and Katie A Burnette . I'll add that the individual BCR and ABL reactions need to be standardized. Try a gradient PCR, with and without DMSO to see if you can get a crisp single band with no smear/ non-specific bands. The chances of getting the fused amplicon will improve if the templates are of good quality.
Jérôme Teulière
Hi Jerome,
Thank you for the input. I'll try this and will get back to you!
Katie A Burnette
Hi Katie,
The expected size are,
BCR - 285bp
ABL - 570bp
The primers were designed to have maximum 60% GC content at ~66c annealing temperature.
We have Simpliamp at lab, hence I used veriflex at different temperature as mentioned above.
Annealing 60c/64c/67c/ 30secs for 35 cycles.
Thanks for the input Katie.
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