difference between cDNA and mRNA - (Sep/23/2013 )

Dear all,

I have to clone a gene which shows "....mRNA, complete cds" on PUBMED.

Now I need to find out the target sequence and design a primer for cloning this gene.

I know I would have to extract the RNA and convert it to cDNA during isolation.

However, can I directly use this "....mRNA, complete cds" to pick primers in blast? Or do I need to pick primers in the mRNA sequence?

Thank you very much for your help.

Basically the sequence you see on pubmed is the cDNA sequence - RNA would have uracil (U) in the place of thymidine (T), and isn't currently sequenceable in the manner that DNA is. 

A forward primer would be the same as the sequence you are seeing on pubmed, and the reverse would be the reverse complement of the sequence you see.

bob1 on Mon Sep 23 07:54:39 2013 said:

Basically the sequence you see on pubmed is the cDNA sequence - RNA would have uracil (U) in the place of thymidine (T), and isn't currently sequenceable in the manner that DNA is. 

 

A forward primer would be the same as the sequence you are seeing on pubmed, and the reverse would be the reverse complement of the sequence you see.

Thanks! I would like to ask, if my forward and reverse primer includes the cds region of the cDNA sequence, is it correct?

Just remember that cDNA has the intronic sequences removed.

helpwithdna on Mon Sep 23 11:05:17 2013 said:

 

bob1 on Mon Sep 23 07:54:39 2013 said:

Basically the sequence you see on pubmed is the cDNA sequence - RNA would have uracil (U) in the place of thymidine (T), and isn't currently sequenceable in the manner that DNA is. 

 

A forward primer would be the same as the sequence you are seeing on pubmed, and the reverse would be the reverse complement of the sequence you see.

Thanks! I would like to ask, if my forward and reverse primer includes the cds region of the cDNA sequence, is it correct?

 

Correct for what purpose?  If you want to clone the coding sequence, your primers essentially need to incorporate the start and stop codons, but if you just want to measure presence/absence then any primer pair should work.

The only caveat to that is whether your gene is subject to intragenic duplication or full deletion.  But that's usually rare and specific to certain genes.