I am in the process of sequencing a gene using Big Dye. I need to complete the ends of the genes using internally binding sequencing primers since I am unable to obtain good sequences at the 5' and 3'ends on the first attempt.
I guess it is comparable to regular PCR - the 3 bases at the 3' end are most important for the enzyme to initiate the reaction. One way to overcome mis-annealing is to lower the annealing temperature a notch - with the risk of getting nonspecific binding of course.
at-least 10-12 bases at the 3' region should be complementary... You can just refer Codehope primer design. They use degenerate primers, it can help you to get an idea about % of complementary region...
Another option is u can perform 3' and 5' RACE PCR to get your targeted sequence
I guess it is comparable to regular PCR - the 3 bases at the 3' end are most important for the enzyme to initiate the reaction. One way to overcome mis-annealing is to lower the annealing temperature a notch - with the risk of getting nonspecific binding of course.
Totally agree with Anandhakumar and Rasmus. Anyway, what are the lengths of the genes you need to sequence? Usually you start sequencing the 3' and 5' of the gene. If the gene is too long you don't have the sequence in the middle of the gene and you need design other primers to continue the sequencing. So, I don't understand well why you have problems in the endings. Maybe you have design the primers very close or just within the ending? Commonly the first bases (10-30) are not readable.
Another option
Generally to avoid 3' and 5' end sequencing error, we used to clone the PCR product in TA vector and sequence it with vector specific primer....
No, they do not! In my experience the most important part is the 5´end. So it will be enough sometimes to have a few matching bases.
I designed primers that went across exon-intron borders at the cDNA and they managed to report sequences of genomic DNA in the intron as well at the exon at the same time. The only problem you can get is that if by chance it binds to other sites you can get a mixture of sequences as result. Then you should modify the primer, or right away you can design socalled degenerate primers that have a mixture of nucleotides at the same position and can bind to more than one possible sequence. You can design as well primers that contain chemically modified nucleotides (like inosin as I remember) that will bind for example to either pyrimidines(C and T) or purines (A and G) only.
If it is possible to subclone your region of interest first, then you basically eliminate the background and will be able to amplify your sequence with a degenerate or not perfect matching primer even more easily.
@ Leopold
The extension start from 3' end, If u say 5' region is important for primer designing could you please explain the logic in that?
Don't take this in a debate way, I just eager to know the new strategies...
Thanks
Kumar
@Anandhakumar
You are right: It´s the 3´end of the primer that I meant! I mixed it up. Thank you for claryfying.
3'-ends of primers should not be complementary (ie. Base pair), as otherwise primer dimers will be synthesised preferentially to any other product;
primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided.
If I am getting it right:
You can use oligo for sequencing even if they are not 100% complementary, it is important to have the 3' end perfectly annealed ( I would say at least 60% of the oligo).
I usually use the same primers for PCR to sequence, in this way I design them at least 40 bases before the point I want actualy to be read by sequence.
Usually you lose the first 10-40 bases from your sequencing oligo because of technical issues. If your amplicon is to big to read the 5' end with the 3' oligo (and viceversa) then you need another set of primers.
I hope this was helpful.
Good luck,
E
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