Urgent! Sequencing a mixture of ssDNA fragments of the same gene
Posted 13 May 2010 - 05:25 PM
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
Posted 13 May 2010 - 06:39 PM
To answer your question... if you took your whole PCR product, including the wrong sized fragments you would not produce readable sequencing results.
Posted 13 May 2010 - 07:23 PM
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?