We're working in a sequencing project of a CYP2D6. Because of some financial drawbacks, we're focusing only in exome sequecing of this gene. That CYP, CYP2D6, has 5 transcripts, 3 of them translates into a functional protein, and, so, we decided to choose first transcript and do the exome-sequencing. Our major doubt is in the moment of choosing the tissue for sample extraction. Will peripheral blood be enough? this sample will contain primarily my first isoform? How could I know?
I would be grateful to receive some advises, also, if you have time to exchange some ideas of this, because we're pretty novice in the topic.
Note: We're not using Next Generation Sequencing, we are gonna use Sanger Sequencing.
Thanks you so much, Michael. I was really scared that the expression in WBC could be not enough to obtain, at least, some amplicons. Now, that we have some data with your help, we have some more self-trust to design the primers for the first transcript. And, as you said, there is no other choice than working with it until we can discover if it's going ok or not.
PS: It's the first time that I use GeneCards site, it was really awesome. Thank you some much, again.
You really confused me with "exome sequencing". That term is usually used when you sequence the entire coding sequences (on DNA basis) of the genome, the "exome". You are talking about simply sequencing cDNA.
Most likely, the main transcript is the long isoform 1 as in NM_000106.5. You need to decide what transcript to sequence whether you go for DNA or cDNA analysis.... When looking at what transcript to analyse, always look at what the literature uses/considers to be the main transcript.
For most genes, you will find at least a little transcript in leucocytes. The bigger issue here is: are the mutations that you expect stable on mRNA? Nonsense and frame-shifting mutations that result in a stop codon before the final exon are prone to nonsense-mediated decay. This issue can be bigger if low-expressing tissue is analysed. Plus in CYP2D6 there are also complete deletion alleles (CYP2D6*5) that you will not see in cDNA.
Specifically for CYP2D6, labs with limited resources (and often "rich" labs as well) usually only analyse a few prevalent mutations. Frequencies of different mutated alleles in different ethnicities are widely published for CYP2D6. So you generally know what mutations to expect and how many you would miss if you limit your studies to a subset of mutated alleles. I would consider this approach over cDNA-Analysis....
Good luck!
Lars
PS: Micheal, note the log-scale of the expression data on GeneCards. Liver expression is about 10 times higher!
I'm so sorry, I couldn't deal with some things on the lab and I've been disconnected from ResearchaGate for a while... ( a long one...). I really appreciate your answer.
Completely true, dear Lars.
It's just a simple sequencing of certain exons of this gene, I confused the terms, my fault. We hope that in a couple of years, we can "evolve" from simple sequencing to NGS.
Now, after 1 month of reading about this topic but focused on other disease, I can understand what you've tried to explain me. It's true, furthermore, I think that CYP2D6 it is polymorphic enough to use sequencing, I didn't do a cost-benefit study yet, but as you said, I must check the prevalence of mutations in this gene taking into account my target population and related population.
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