An RNA question !!!!***

polk at molbiol.ox.ac.uk polk at molbiol.ox.ac.uk
Wed May 11 05:10:55 EST 1994


In article <2qn6urINNfs1 at dns1.NMSU.Edu>, smori at nmsu.edu (Shahram Mori) writes:
> 	I have an Unknown RNA molecule that I would like to Liagte
> 	to a vector. T4 RNA ligase can use the 5'P overhang of a 
> 	rescriction enz digested- DNA as a donor to ligate DNA-RNA.
> 	This allows an RNA molecule with 3'OH to be ligated to a
> 	ds DNA. I plan to use RT to make a cDNA copy.
> 
> 	*************
>    DNA	***************5'P  +     OH3'----------------------5'OH  RNA
> 	
> 	Make the first strand cDNA synthesis.
> 
> 			cDNA
> 	*************=================
> 	***************P-O------------5'OH
>   	
> 
> 	Do PCR:
> 
> 
> 	************=================3'OH
>         <------------------------||||(primer compl. to cDNA)
>    Vector spfc|||------------------>
>       primer
> 
> 	
> 	And then use Dideoxy sequencing to get the gene.
> 
> I would like your comments on this method.Has anybody done an RNA ligase
> RNA/DNA ligation and are there any tricks to it? Also since the sequence
> of the RNA is not known is it best to use the a random Hexamer to get a
> complemenatry sequence to the cDNA for the PCR?
> 

Hi Shahram Mori,

Have you seen the paper by Hetzer and Mueller in Nucleic Acids Research 1993
vol 21;5526-5527: PCR mediated analysis of RNA sequences.

They outline exactly what you are thinking of doing. I haven't tried this 
myself yet though, just grabbed the technique because it looked useful :)

Best of luck
Ian 

*******************************************
Ian Polkinghorne (polk at molbiol.ox.ac.uk)
NERC Institute of Virology
and Environmental Microbiology
Mansfield Rd  Oxford   UK



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