Hi,

I never did that to reduce the number of sequence reactions. You should think that there is also a limit in the size of the fragment you can read by Sanger sequencing. If you are able to set up the PCR conditions for your 14 exons easily, then it is not too much work, just organize them in plates and would be around 4 plates/exon. Maybe it's useful for you to have also the flanking regions of your exons.

Other option could be a high throughput technique, as ionTorrent sequencing, with which you can sequence certain region of a genome all at the same time. Allways depends on your budget...

hope you can go ahead!

I have only the total DNA. 

Hi Nguyen,

If you don't have option of using RNA or cDNA ...you can design a strategy to amplify the exons separately and clone multiple fragments in the vector...there are some vectors also available that can handle multilple fragments and even without using restriction enzymes. Or else you can also design a strategy of your own...as there are 14 exons and you need to sequence them in 400 samples...you can design 28 primers to amplify them and can use the approaches mentioned above to ligate them and clone collectively for each sample and sequence in a single reaction...using sequence analysis tools you may need to to do some editing later to organize them properly....since you have limited options with your samples you may have to work using these type fo approaches.. 

The ideal and straighforward option could have been using the mRNA and sequencing the cDNA of the genes from all the sample..

bye

Thank you very much Ajay Saini for your help. Actually i'm thinking about design the pair of primers that amplify 1 exon and those primers have the homologous region with the other pair of primers that amplify 1 other exon near by. Thus i will have a bigger PCR product (= total size of 2 exons) and i can decrease the number of sequencing reaction i have to perform. But there are some experimental question like: can i perform PCR with all primers in the same tube or put them separately, and after do i need to add some enzyme like T4 ligase to connect the PCR products... I will be very thankful if someone can give me more details,... or a protocol if it's possible.

SIncerely

Hi,

I never did that to reduce the number of sequence reactions. You should think that there is also a limit in the size of the fragment you can read by Sanger sequencing. If you are able to set up the PCR conditions for your 14 exons easily, then it is not too much work, just organize them in plates and would be around 4 plates/exon. Maybe it's useful for you to have also the flanking regions of your exons.

Other option could be a high throughput technique, as ionTorrent sequencing, with which you can sequence certain region of a genome all at the same time. Allways depends on your budget...

hope you can go ahead!

Many thanks Bàrbara. All exons of my gene are quite short (around 150bp) so i think it could save time if i can join 2 or 3 exons together but may be you're right. I should go straight up, sequence one - by one. Your idea about ionTorrent sounds really promising.

Thank you all Seema; Ajay and Bàrbara for your help.

An efficient solution is to design a custom targeted enrichment oligo set (you can easily do that with say Agilent or Illumina), comprising all the exons, to pull-down the targeted regions and to sequence them with an HiSeq. Given the relatively small size of the region (just a few exons), the overall NGS sequencing cost should be reasonable. If HiSeq sequencing is not available at your institution, there are many companies offering this service and, although generally speaking it is not cheap, I suspect that sequencing all the exons one by one will be also quite expensive.

Thank you very much Rocco Piazza for your suggestion. It's really helpful

You can try to use the high resolution melting analysis.

http://www.lifetechnologies.com/de/de/home/life-science/pcr/real-time-pcr/real-time-pcr-applications/genetic-variation-analysis-using-real-time/high-resolution-melting-hrm/mutation-scanning.html

Hi Nguyen, I think you have already some good suggestions in prev. answer. We are encountering same questions as you have and most of the time the answer is: Sangersequence all the exons in the gene. I looked at NDST1, and noticed that it have intronic regions that are typically several hundred bp in lenght. This would not favour making amplicons that contains two, or more exon. If you intend to do even more sequencing of NDST1 tahn these 400 samples, then you might wnat to use a NGS-approach, targetted sequencing like AmpliSeq or other. But, this will requrie much more from you when it comes to setting up the analysis and taking care of the data, both in time, experience, and cost. Hope this will help you to choose wisely ;-)

Thank you Alexander Zink and Hakan Thonberg for your suggestion. With the NDST1 gene, the exons are quite small (some less than 100bp) and the introns between exons are quite large so i still prefer find some techniques that could amplify and then connect the exons - and then: Sanger sequencing. I'm currently reading about a technique called "Overlap extension PCR". It's can help to amplify 2 exons from a distance, remove the intronic DNA between them so i think that this technique could help me. This method seems quite easy and cheap too. Does anyone who has performed this technique can give me some advices? (or a protocol perhaps)

Thank you all!