I want to study gene rearrangement(fusion) of ALK, ROS1 and RET genes in lung cancer at gDNA level. What is the best possible way for it?
Is it fine to make primers specific to fusion area and then go for PCR and is amplification occur it means its positive and then confirm by sequencing.
Looking at fusion genes at the DNA level can be difficult. Because the fusions occur in introns, and sometimes even multiple possible introns in each participating gene, the recombination events can happen over a large region. You would have to use PCR primers to tile the region in PCR reactions to make sure that any products are in a reasonable size range and this may be impossible to do exhaustively. You may also have to put in a lot of effort on various artefact bands that come up. There is additionally the problem that you can get PCR artefact bands from chimeras formed with repetitive DNAs near your primers that join together in the PCR reaction. The most robust fusion assays are carried out at the RNA level, where you can put the PCR primers in exons that should be included and the size of the product is reasonable.
As an alternative approach, I would consider developing a high density, custom array across your potential recombination regions. You can then either use a microarray hybridization approach to estimate the junctions based on CNVs in the area (the Lupski lab has published several recent papers showing the power of this). Alternatively, you can use the array for hybrid selection of next generation sequencing libraries and then utilize bioinformatic approaches to identify the discordant read pairs to track down the exact junction. The problem with these latter approaches is that the cost is in the hundreds of $ per assay.
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