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RT-PCR primer


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#1 AdioZAmigoZ

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Posted 16 March 2011 - 06:16 AM

Hi everyone.
I want to know the difference between a forward primer and a reverse primer.

5--------------------------3
_____________3--5 reverse primer

__5--3 forward primer
3--------------------------5

When I look at the upper "picture" (try to ignore the "____" I just had to use them to move the primers the their place :P) it seems to me both are the same. They both goes "towards" 5 on their DNA string.

I also have a question concerning whether you use a reverse or forward primer for RT-PCR on mRNA. I read it was a reverse primer but I thought myself it was forward primer.
In our cDNA there was a sequence of bases which was exactly the same as that of our foward primer.
So I thought that we had used forward primer on mRNA and thats why the primer had been part of the created cDNA.
Thanks in advance.

Edited by AdioZAmigoZ, 16 March 2011 - 06:19 AM.


#2 pcrman

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Posted 16 March 2011 - 08:26 PM

I am not sure if I understand your question correctly. The two primers do not bind to the cDNA sequence at the same time because RT reaction creates single stranded cDNA that is complementary to the RNA sequence. In the first round of PCR, it is the forward primer that first binds to the template, after the first round reaction, a complementary strand of DNA is synthesized and then the reverse primer binds to it and initiate the synthesize of additional strands of DNA.

I guess your 2nd question is whether you use forward or reverse primer for RT reaction (not RT-PCR), correct? The answer is: reverse primer. It is the same as you would use Oligo-dT primer which binds to polyA tail in RT reaction

#3 Nephrite

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Posted 17 March 2011 - 03:09 AM

I am not sure if I understand your question correctly. The two primers do not bind to the cDNA sequence at the same time because RT reaction creates single stranded cDNA that is complementary to the RNA sequence. In the first round of PCR, it is the forward primer that first binds to the template, after the first round reaction, a complementary strand of DNA is synthesized and then the reverse primer binds to it and initiate the synthesize of additional strands of DNA.

I guess your 2nd question is whether you use forward or reverse primer for RT reaction (not RT-PCR), correct? The answer is: reverse primer. It is the same as you would use Oligo-dT primer which binds to polyA tail in RT reaction


Hello pcrman.

I am totally confused now.
Of course, I know that cDNA is single stranded.
BUT...
I do two-step RT-PCR, i.e. first I do the RT reaction, and then - conventional PCR.
I use RevertAid M-MuLV Reverse transcriptase from Fermentas - in datasheet they say that the enzyme has RNA- and DNA-dependent polymerase activity and an RNase H activity specific to RNA in RNA-DNA hybrids, i.e. 3 in 1. RNase activity cleaves the RNA, and DNA-dependent polymerase builds the second strand.
Thus, I believed that in the end of reaction I have double stranded cDNA.
Am I totally wrong?!

And.....you use specific primer if you want to acheive cDNA of a particular transcript, right? For total cDNA you use random hexamer primer, is that correct?

Nephrit

#4 seed

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Posted 17 March 2011 - 08:42 AM

I have extracted both gDNA and RNA from tomato seed. Then for my RNA, I did the DNase free treatment, running the gel (the bands are cleared),and synthesized my cDNA. And then I ran PCR together for both of my cDNA and my previous gDNA,using the same condition for both cDNA and DNA. After I ran my electrophoresis, only my housekeeping genes (for both cDNA and DNA)and my DNA showed the bands and no band present for my PCR product from cDNA. I think my cDNA is okay since that my housekeeping gene was working and my primer is okay too because my PCR product from DNA showed the band on my electrophoresis. Can someone explain to me why this is happen?? ASAP....




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