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Urgent! Sequencing a mixture of ssDNA fragments of the same gene


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#1 phucvn

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Posted 13 May 2010 - 05:25 PM

Hello,

I amplified a gene from my cDNA using a specific primer (sure this primer works). I got a mixture of fragment of different sizes. I suppose that may be the template consists of different short and long copies of that gene. I want to sequence these ssDNA copies (using the same specific primer in PCR) to reach the terminal ends of the gene. But I am wondering a question:
Does it result in a readable sequence when sequencing a mixture of ssDNA amplified with the same primer?
If it does, that's good for me. Suggestions, please
Thanks

#2 Micro

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Posted 13 May 2010 - 06:39 PM

Having different length fragments in your PCR product may just be non-specific primer binding. Do you have a band at the expected length? How many copies of the gene in the genome and do you expect each copy to be different? If you have the correct length present and a gene with only one type present in the genome, then I would cut this correct sized band from the gel, purify and sequence this fragment.

To answer your question... if you took your whole PCR product, including the wrong sized fragments you would not produce readable sequencing results.

#3 phucvn

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Posted 13 May 2010 - 07:23 PM

Thanks, Micro for your quick comment.

I am sure that my target gene has only one copy in the genome. The difference between fragments I mean here is in length because i) I extracted RNA from whole larvae and mRNA I got may be broken or by several reasons during the process that it would not in the same size; ii) PCR product derived from cDNA is in different length because I run PCR with only a single primer, so ssDNA different in length will be amplified. When I used both fwd and rev primers I got only a single band.
Previously, from this band I sequenced it and got a specific read but not enough to make RNA probe. So I want to get longer reads up to 3' and 5' termini of the gene without doing RACE which is very expensive.
How do you think?




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