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Molecular bio novice, a little help?


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I'm trying to figure out how to sequence a gene I'm interested in from mRNA.  Typically the way the lab does qrtPCR is they first make cDNA.   using Applied Biosystems high capacity RNA to cDNA.  Does this create full length cDNA of every RNA?  For example, one gene I want to study is 5000 bp long in its mRNA.  Does the reverse transcriptase make a full length cDNA?  Next, what do I do with the cDNA?  If I design primers using standard protocols that use online software, it gives me a pair of primers that are somewhere in the middle of the cDNA.  Once I do PCR, will it amplify the whole cDNA or just the portion between the two primers?  The problem is that I want the whole cDNA sequence to be amplified and sequenced.  The entire sequence is already known for the mRNA, if I just designed primers that work all the way at the end of the 5' and 3' ends, would I get the entire sequence amplified using regular PCR?  Would it have trouble amplifying a mRNA with 5000 bps?  Would RACE PCR be appropriate here to create the entire cDNA amplification or is it not needed since the entire gene sequence is known for cDNA so I can get away with just regular PCR? Thanks in advance for any help.

 

The reason I want to sequence the whole cDNA from mRNA even though the sequence for cDNA is already known is to look for mutations.  

Edited by fibonacci
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This probably isn't the best forum for technical questions, but oh well.

 

Most of the cDNAs will not be full length. If you've just got a kit with random hexamer and oligodT primers, you'll have a mix of "all" cDNAs. But you could amplify selectively too if sequencing a single gene is your goal.

 

If you need to amplify an entire gene, don't just use some random primers generated in the middle of the reading frame (was the web app doing it for qPCR?). As you seem to suggest, you'll only get the piece in the middle. 5000 kb is an extremely long PCR. There's no reason you can't just do it in pieces and sequence those. Unless you're using a polymerase like pfu or phusion, you're going to introduce mutations when amplifying, which would be counterproductive for your purposes.

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I've never had a problem amplifying 5kb constructs with Pfu polymerase like virion suggested. I don't think you'll be able to sequence anything close to that length though. If you already have the mRNA sequence, why are you sequencing the DNA? Just a confirmation that it hasn't mutated?

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I think I was trying to help troubleshoot the problem without actually thinking about what was being done.

 

Why sequence the RNA (cDNA)? If it's simply point mutations you're looking for, sequence the genome. Only reasons I can think of would be if it's an RNA virus you're sequencing, or if you're looking for splice site junctions or something.

 

Mistakes in transcription happen, but they don't happen clonally. Meaning, a few copies of mutant protein could be made but not in a way that would affect the system you're interested in. You want to look at the genome. I'm not sure if even deep sequencing techniques would pick up single mRNA variants (would be neat, though), but Sanger sequencing would certainly be useless in this task.

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