I want to sequence RAPD fragment by cloning it. I don't know if my fragment of interest is homologous and it's quite possible that few non-homologous fragments of the same size migrate together. Is there a way to find out how many of them I have and how to separate fragments of the same size but different in their sequence, so I could get them all cloned and sequenced? thank you for the answers.
SSCP is told to separate molecules according not only to size but to sequence. It is a bit complicate, as it usually involves acrylamide electrophoresis and denaturing gels. I have never recovered bands from acrylamide gels, so I can't help you on this subject. Anyway, bad sequencing results with plenty of multiple peaks is a clur for indels or for mixed molecules in your band...
Dear Artur, I was thinking to clone and then to check colonies by PCR, but if there is one fragment in very low copy number - it might be missed, and also it seemed to me very laborious, if I get most colonies different, but the main reason to ask was to get more opinions in addition to mine, 10 brains are always better than just 1.
Thank you for the answers, and may you all have nice and warm Christmas and a Happy New Year!
Hi Asta,
Basically, I fully agree with Artur Burzynski and Ahmed Mansour Alzohairy. Cloning might be the best choice. You just need to sequence sufficient number of clones, which is not more laborious than SSCP.
Although, if you want to try other methods, SSCP and heteroduplex-analysis could be recommended. However, if a particular fragament has got a very low copy number, these methods might not be sensitive enough.
I have a question... how could you know that the fragments you have are non homologous based on size only? To decide, you need to seq. them... and to seq. them you first need to use regular electrophoresis/cloning methods...
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