Hello everybody,
I have a question to chromatograms.
Is it possible to determine whether a tumor mutation is heterozygous or homozygous when DNA from tumor cells is isolated and amplified by using PCR and sequenced?
I've read that if the mutation is heterozygous a single peak position within a trace may have but two peaks of different colors instead of just one and that this was common when sequencing a PCR product derived from diploid genomic DNA, where polymorphic positions will show both nucleotides simultaneously. (The University of Michigan DNA Sequencing Core Interpretation of Sequencing Chromatograms https://seqcore.brcf.med.umich.edu/sites/default/files/html/interpret.html)
The DNA which is analysed origins from several tumor cells so the chromatogram presents an average of the tumor cells, doesn't it? So how can the chromatogram make a statement about whether the DNA mutation of a single tumor cell is heterozygous or homozygous? It sounds more logical for me that it states that when I have a double peak I can just say that I have DNA strands in the tumor cells that differ from one other and the tumor is heterogenous but how can it be indicative of tumor cells being heterozygous or homozygous? Couldn't it be possible that on the one hand the tumor has cells that have the mutation and on the other hand there are also tumor cells that don't have the mutation?
I'm looking forward for answers and thank you very much.
Assuming that you are referring to Sanger sequencing then you are always going to have problems if the template is mixed prior to analysis and generally both of your hypotheses are possible. In some cases it may be possible to be more precise. If in the sequencing you find that there is a nearby polymorphism in your cell line dna then it may be possible to associate the mutation M with one allele of the polymorphism ( let us say that is base A). Then if you ARMS amplify the A allele in your sample and sequence the product then if the product only has the M base then you do not have a mixture of tumour which has both M and non-M ( cloning would also give this information ). NGS would be more informative. In some cases your example would be even more complex as some normal cells will also me mixed in with the tumour cells
The short answer is, no you can't tell if a single tumor cell is homozygous or heterozygous in a mixed sample from many cells just by Sanger sequencing. You can detect SNPs, small changes in homopolymers length, etc. by Sanger, but it is NOT the preferred method. NGS is a better approach.
One important point to consider is the purity of the sample. In tumors, you always have contamination by normal cells (infiltrating lymphocytes and so on). As long as you do not have this info, you will not be able to define the status of your mutation
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