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Reverse transcription of a circular RNA template

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Reverse transcription of a circular RNA template

Postby frankalpha » Mon Sep 20, 2010 3:41 pm

As above, will the cDNA product be linear or circular (say if I use Superscript II or III)?
I suppose the cDNA product to be single-stranded. Am I right?
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Postby JackBean » Mon Sep 20, 2010 4:18 pm

The cDNA will be circular only if you use ligase and it will be dsDNA, if you use primers for both strands of course. However, if the primers will be far enough from each other, you will get broken cirkular dsDNA ;)
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Re: Reverse transcription of a circular RNA template

Postby frankalpha » Mon Sep 20, 2010 5:52 pm

Thank you for your reply.
I use only one primer for the reverse transcription and there is no ligase added, so the cDNA should be single-stranded and linear, right? If I want to get dsDNA, can I use DNA Polymerase I to extend the other DNA chain, using the hairpin loop at the end of the first-strand cDNA as the primer?
I'm a little confused about your 2nd sentence though. Are you talking about a RT-PCR?

JackBean wrote:However, if the primers will be far enough from each other, you will get broken cirkular dsDNA ;)
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Postby JackBean » Mon Sep 20, 2010 9:05 pm

And where will you get the hairpin?

You're not talking about RT(reverse transcription)-PCR?
http://www.biolib.cz/en/main/

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Re:

Postby frankalpha » Tue Sep 21, 2010 1:57 pm

JackBean wrote:And where will you get the hairpin?

You're not talking about RT(reverse transcription)-PCR?



No, I'm not talking about RT-PCR, just RT instead.
The hairpin theory came from this link 'http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm'. The 2nd paragraph and fig. Thank you very much.
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Postby JackBean » Wed Sep 22, 2010 11:56 am

well, so you're interested in 1st strand synthesis only, rigth? But then you will really get linear ssDNA
http://www.biolib.cz/en/main/

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Re: Reverse transcription of a circular RNA template

Postby frankalpha » Wed Sep 22, 2010 9:05 pm

Actually, I want to get ds cDNA from RNA. I've figured it out. There are several kits available that can synthesize ds cDNA from RNA (there is a step for second-strand cDNA synthesis after the first-strand synthesis). Thank you for your help.
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Postby JackBean » Mon Sep 27, 2010 9:03 am

OK, nice, but how do you get the hairpin? You had to have some self-complementary region in the mRNA, hadn't you?
http://www.biolib.cz/en/main/

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