I have a viral linear DNA sample extracted from an ocular washing solution.
I want to run sequencing for its two genome ends (under 1kb each and I have known the sequence of the rest of its genome).
If I design 01 primer for each end, would it possible to run sequencing on teamplate which is a DNA extracted sample?
Or anyone can suggest other ways to sequence the genome ends of a linear sequence?
Thank you all.
Hi Tuan,
You can clone the genome into a vector of your choice (depending upon your restriction sites selected). Then you can confirm the sequence with the 5'-end and 3'-end primers of your vector. Also sequence it using your Insert primers. Good Luck!
Hi Tuan, your primers will bias your results for your sequencing. I would suggest doing RACE or DNA circularization to determine the genome ends' sequence. I believe you can get RACE kits or modify the attached paper's protocol for your virus. For DNA circularization you could follow the below paper. I will caution though, the DNA circularization is a little hard to do, particularly if you have a large genome. Also, you would need a lot of starting material, so maybe your best bet is RACE.
DNA circularization paper - https://www.nature.com/articles/srep24722
Good luck!
Joyce
Hi Tuan,
You can clone the genome into a vector of your choice (depending upon your restriction sites selected). Then you can confirm the sequence with the 5'-end and 3'-end primers of your vector. Also sequence it using your Insert primers. Good Luck!
Hi Tuan
I also suggest cloning and sequencing approach as stated by Pratibha Srivastava.
Best wishes
I also think cloning into a suitable vector and then sequencing might be the easiest option
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