Sanger Sequencing DNA fragments

Go to the profile of stilldontknow
Jan 07, 2018
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Hi,

I am a PhD student, doing work mainly on bioinformatics, thus my simple question that follows.
For my work I will need to perform Sanger Sequencing to confirm the presence of multiple different SNVs in a sample of individuals with a given disorder.
To confirm a single SNV by Sanger, I first have to amplify a DNA fragment, from the genomic samples of the individuals, which will be done by conventional PCR. For the sequencing itself, where we expose the obtained fragment, separately, to forward and reverse primers, is it correct to use the same pair of primers I used for the conventional PCR? Since the region complementary to the primers will still be in the fragment, I suppose this is ok.

Sorry for my really basic question.

Thanks in advance

3 Comments

Go to the profile of mdfenko
mdfenko 5 months ago

you can but...

you really want primers that are in from the ends and sequencing primers are usually longer than amplification primers so that they will be more specific and less likely to slip.

Go to the profile of OldCloner
OldCloner 5 months ago

Hi Stilldontknow- you’ve hit on my “cup of tea!” mdfenko is correct in that primers set in from the ends have the advantage of increased specificity. Meaning if there are any faint, non-specific amplicons resulting from your reaction, internal sequencing primers will ignore them and only stick to the proper amplicon. However, if your amplifications are very clean, often you can get away with using the amplification primers for Sanger sequencing. (I do it all the time). Consider your primer Tms- those between 50 and 60 degrees C are best for sequencing, but higher ones can work. Avoid using degenerate primers for sequencing.

You must purify your amplicons to get rid of any left-over amplification primers first. I recommend a mini-column purification (many companies make similar kits) so that when the sequencing tech adds sequencing primer, there is only ONE in the reaction. Also, this allows accurate quantitation of your purified product, which is very important in in sequencing PCR fragments. I would avoid gel-purification if possible.

Also-how large are your amplicons? Fragments less than about 150 bp don’t sequence well, at least in the dye-termination type sequencing reactions I am familiar with. Try to make your primer sites upstream from the variable nt positions by at least 100 bp, for a real clear read.
Good luck!

Go to the profile of Daniel Tillett
Daniel Tillett 2 months ago

One solution is to clone the PCR products and then sequence a number of clones. You can even be really tricky and just PCR the ligation mixture with the vector primers and then use the PCR amplification primers to sequence the fragment. I have done this before in the past when I need to sequence in close to the original PCR primer location on a fragment that was too long to sequence from the reverse end.