Hello :)
I am looking at the presence of toxin biosythensis genes in lake sediments. Nothing fancy, since I only want to verify the specificity of PCR results for primers related to marker genes for cyanotoxins biosynthesis (e.g. mcyE for microcystins); I am using the Sanger results to confirm which organism in a given sample is responsible by searching for the results in BLAST.
I have so far done both forward and reverse Sanger, created a consensus sequence after trimming and then run the search. I can see the benefits of having the Sanger done both ways for some samples because one or two stopped abruptly, so then I could rely on the opposite read and a few bases had low quality scores.
But beyond quality control and upping my chances of getting a longer read, is there any benefit for doing it twice for a fairly simple "whose genes are these?"/PCR product verification approach?
Thanks :)
If you need the sequence to show the identity of 2 or more similar genes then forwards and reverse is ideal but if the question is "does this sample have my gene" then sequencing in one direction and blasting 120 bases and getting 99% identity is good enough to say "my sequence exists in my sample" and adding another 400 bases probably does not tell you much more useful information and save doing the reverse sequence for the few samples that look like indels or repeat sequences
I completely agree with Paul Rutland.
However, I would like to add another point. If you only want to check the presence and absence of a particular gene. There is no need to go for sequencing which rather increases the cost of the assay. Instead, you can opt for specific primers (or design one based on previously published gene sequences) and check whether it amplifies in a sample. Depending on the presence or absence of the amplification product, you'll get confirmation without sequencing the amplicon.
Thank you both for your answers :) It's very helpful.
Gutha Ramesh we are doing the sequencing to determine which bacteria is responsible for the synthesis of the toxin, so we need more than just the band in the gel since the sequence can tell us whether the toxin was made by bacteria a, bacteria b, or c etc... due to differences within.
So the question is more like:
'1 does the sample have the gene?',
'2. Ok, so whose gene is it?'
Single site (either Forward or reverse) sanger sequencing is enough to know about the sequence of gene.
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