Direct DNA sequencing - sequencing without PCR product (Dec/02/2005 )

Hi all!

I could need some advice concerning direct sequencing from DNA.
My gene of interest is damaged on genomic level and is probably fused to an unknown sequence.
I can get a PCR product for certain areas but in 3' direction their is an aberration for sure as I can't get PCR products.
I'm through with 3' RACE but would need to know more for the genomic level.

I want to avoid inverse PCR or something because I don´t really have an idea what´s going on in the 3' end here. I therefore thought about direct sequencing from genomic DNA without performing a 'normal' PCR before.


Has anybody of you ever did that and can give me some advice??

Thanks alot for any input.
Cheers

I've done it before, but only with Mycoplasma, which have a very small (800Kbp) genome. There is a paper by Cheryl Heiner "Sequencing multimegabase-template DNA with BigDye terminator chemistry" Genome Research 8(5):557-61. I ignored the thermofidelase enzyme suggestions, but cycled 100 times as they suggested. It worked, but the signal was quite noisy. The sequence was good enough to derive a primer, which I used for PCR and resequenced the region.

Hi!

Thanks a lot. I´ll check that paper you suggested. In the meantime I found a paper which is about 'suppressor PCR' technique; this uses adapter ligation after retriction digest of genomic DNA. Only fragments that contain a gene specific primer is amplified by PCR (well, at least in theory). I think I check that in parallel.
Thanks a lot for input.
Cheers

I have used this technique before with a lot of success. I think the author for the paper is Siebert. A colleague in our department added a couple of short cuts such as doing the digestion and ligation at the same time (overnight at 20 degrees) and then using 1 ul of the reaction directly into the PCR. No purification steps required.

[quote name='ML1975' date='Dec 3 2005, 10:21 AM' post='33324']
I have used this technique before with a lot of success. I think the author for the paper is Siebert. A colleague in our department added a couple of short cuts such as doing the digestion and ligation at the same time (overnight at 20 degrees) and then using 1 ul of the reaction directly into the PCR. No purification steps required.


Hi!

Yip, you are right the first author of the paper is Siebert. I hope I can adapt it.
Do yo remember if you had to change things or did you follow each step of the paper first and were already successful?
Any advice highly appreciated. Thanks again.
Cheers

If you follow the procedure as its written in the manuscript it will work. The changes that we made were just to make it quicker, just shortcuts. We used one-phor-all buffer and did the ligation and digestion in the same tube at the same time to save time. You may need to try a few different blunt end cutters before you get a good sized product.

[quote name='Bomber' date='Dec 3 2005, 09:31 PM' post='33327']
[quote name='ML1975' date='Dec 3 2005, 10:21 AM' post='33324']
I have used this technique before with a lot of success. I think the author for the paper is Siebert. A colleague in our department added a couple of short cuts such as doing the digestion and ligation at the same time (overnight at 20 degrees) and then using 1 ul of the reaction directly into the PCR. No purification steps required.


Hi!

Yip, you are right the first author of the paper is Siebert. I hope I can adapt it.
Do yo remember if you had to change things or did you follow each step of the paper first and were already successful?
Any advice highly appreciated. Thanks again.
Cheers
[/quote]

I will give it a shot. As soon as I have some results or more questions I´ll post it.
Thanks a lot for your help.
Cheers