sequencing gene more than 10kb

[genefinder]

Hi All..!!!

I am a new member so kindly accept my greetings and best wishes to all. I have a gene fragment of size more than 10kb and is AT Rich. If i start sequencing by the traditional way i have to design many internal primers and it would be a highly time consuming.

Can anyone help me suggesting me with some new technologies(next generation sequencing) available in the market? Southeast asian services would be a better option. Do I need to clone my gene? I guess i need to as I am working on field isolates and should avoid multiple clones. Please suggest

[krobison]

None of the new technologies will be very cost effective for a single 10Kb gene nor are they likely to be quicker than primer walking. Benefits won't really kick in until you are 100kb or bigger.

[genefinder]

Hi.....

Thanks for your advice(krobison). Well, my sequences wont be more than 10-15kb. Anyways, do you know any other strategy other than primer walking for sequencing this kind of fragment? Suggestions from all members are most wellcome. thanks

[krobison]

The standard old throwback is nested deletions -- digest in from one end with exonuclease, blunt the end, ligate. Pick clones from different digestion times.

Another alternative would be to digest your insert with a frequent cutting enzyme & put those fragments into vectors. Sequence each fragment. Now synthesize primers from near the ends of each fragment; the sequences from these will tell you how the pieces go together.

Primer walking tends to be the favorite because all of the labor is semi-automated; either of the approaches above involve a bunch of additional steps.