I am trying to sequence a 70bp PCR amplicon by Sanger Seq., using the PCR primers as sequencing primers. Will the small size of template be a problem? I tried it once, no result. Should I clone it in to a plasmid first or will it be possible to sequence this 70 bp amplicon directly?
Thanks all.
I might need to know a bit more about what conditions you used but in principle it should work. The small size of the fragment might allow some self-annealing but if you fiddle around with the mass of the primer upping or lowering it relative to the mass of fragment just in case there is a little too much competition between primer annealing and fragment self annealing. I'd also try both primers separately in case one of them has some hidden secondary structure or another non-obvious problem. It's probably something that will just need a little fiddling with. Hope that's of some help!
You should ligate the small PCR products with the cloning vector. Then the universal primer could be used as the sequencing primers
Hi
Using a PCR primer for sequencing small amplicons would never work, because sequencing primers are always designed at lest 50bp away from the start of the original sequence. This is because the sequencer gives only N's before it gets stabilized to give you actual nucleotides and in this process it eats away these 50nts.
The best option is to clone it (TA vector would be easy if your amplicon has an A overhang), then send it to sequencing with a standard primer(T7/Sp6). ..
Thanks Afzal, The sequencing guys told me the same. I am going to clone it in to a TA vector now. Cheers!
Thanks Yunxiang for your suggestions.
David, I was already doing both primers separately, also tried sequencing protocol for high GC content templates, but no result. I guess the main issue is size of the template.
Thanks
Hi
There is another method: you must modify the primers by adding the M13 forward and reverse primers upstream your PCR primers (these modifications are usually proposed by the manufacturer), then the sequencing is performed using the M13 primers.
Choose the best way for you
In principle there is no problem to sequence a short fragment. In practice, if you send it out to companies, it will not work. As already correctly said, the first 20-40 bp are normally covered by the large gaussian of the excess fluorescently-labelled ddNTPs that are not incorporated in the sequencing, unless you precipitate the DNA after the thermosequenase reaction in 2 M NH4OAc (which companies never do for time reasons) or you pass the reaction through a membrane with cut-off size of ca. 3000 Da, such as the Millipore Montage seq. clean-up system (which companies never do due to its costs!). Same problem you would have by adding a universal primer in front of your primers, with the additional inconvenience that then, even if you sequence and it turns out to be ok, you cannot clone it. So, in the end the TA cloning is the best (or the TA blunt if you are using the Pfu for the PCR, which does not add A overhangs...).
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