could you clarify your question a little bit please? - If its inferred from homology, that doesn't mean that its a mouse sequence, it just means that there is an annotated mouse homologue, but no direct evidence of expression from human. so basically you already have the sequence.

unless I misunderstood.

But as a general answer, if you want to sequence a gene, you need to design primers and amplify your target by PCR, then sequence the product. if you do not have the facilities, there are companies that will do it all for you, e.g. macrogen in S. Korea. you will probably still have to design the primers yourself.

Hi Amr,

Thanks for the reply. In Mouse the gene has an evidence of expression and the sequence has been derived experimentally. From that time they derived by homology the sequences for other organisms.

Like i said , i have the sequence which was inferred form mouse based on homology. I have the PCR product by have no idea how to proceed further for the sequencing.

Hope i clarified.

cheers,

Aravind

you can put your PCR product in an envelope, and send it by fedex or something similar to a sequencing service. I have used Macrogen before and they are very good. takes about a week or so to get your results.

http://www.macrogen.com/eng/index/

im sure there are alternatives.

they will email you the results. you would have to specify if you want them to purify your sample, or else do it yourself. They also prefer a certain concentration. the website is quite informative.

We sequence PCRs routinely just to validate the product. Troublesome products (and PCR amplicons less than 100bp) are usually cloned into a TA vector (e.g. pGEM) and subsequently sequenced. That said, this approach validates ONLY the PCR product and not the gene. For validation of the gene you would need to map transcriptional start site and the site(s) of transcriptional termination / poly-adenylation. This is commonly done by 5'- and 3'-RACE (rapid amplification of cDNA ends) for which several kits are available.

Aravind,

If you have your PCR product you can do a gel extraction (if you haven't already) and then prep it for sequencing.

Typically you will need to dilute the your PCR product and primer sets to whatever the company specifies.

For instance we use Genewiz and there specifications for sequencing can be found here:

http://www.genewiz.com/public/Sample-Submission-Guideline.aspx

OVERVIEW

1) Identified your gene

2) Develop or find literature primers for your gene

3) Run a a PCR

4) Extract the PCR product (cut out the band or peck it out)

5) Gel extract the product

6) Prep product and primers for sequencing based on your companies protocol

7) Submit the PCR product-Primer mix

Hi All,

Many thanks for the suggestions. I will consider all of them and discuss with my supervisors which will be the feasible way to proceed. Will let you know about the results as well :).

Have a nice weekend.

cheers,

Aravind

Aravind,

if you have the mouse gene sequence you can BLAST it in the human genome and retrieve everything you need. You may see many features of the gene in NCBI, Ensembl, UCSC genome browser, as well as in GEO and Encode for regulation.

cheers, alessio

i agree with Alessio,

check 1000 genomes, BLAST, etc...

make sure that major sequencing centers haven't done your work for you. in 2013, you should be able to get reference gene structure (i.e. genomic organization and sequence) from databases.

save your wet-lab efforts for studying varying in specific patient samples / specific tissue sources.

Good luck!

John