An RNA question !!!!***

Martin Leach leach at mbcrr.harvard.edu
Tue May 10 06:48:19 EST 1994


In article <2qn6urINNfs1 at dns1.NMSU.Edu>, smori at nmsu.edu (Shahram Mori)
wrote:

> 
> Dear Moleculentters,
> I would very much appreciate your opinion regarding the following
> experiment that I would like to do:
> 
> 	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.

Beware of concatamerisation upon ligation.

Make the other end of the vector DNA blunt/incompatible with the cut end of
the vector where the RNA is to be ligated.

> 
> 			cDNA
> 	*************=================
> 	***************P-O------------5'OH
>   	
> 
> 	Do PCR:
> 
> 
> 	************=================3'OH
>         <------------------------||||(primer compl. to cDNA)
>    Vector spfc|||------------------>
>       primer


If it is an unknown RNA how can u make the primer compl. to cDNA.
If u do not know the RNA - what about it's size. If too big u will not be
able to do conventional PCR. Look into the Barnes method (PNAS) for long
PCR.
> 
> 	
> 	And then use Dideoxy sequencing to get the gene.

Why not clone the whole mess!

> 
> I would like your comments on this method.Has anybody done an RNA ligase
> RNA/DNA ligation and are there any tricks to it?

In the SLIC method (see clonetech kit or various papers (refs not at hand)
RNA ligase is used to ligate a DNA oligo to RNA (i think).

> 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?

Why not
1. remove the cap structure at the 5'end of the RNA
2. ligate an aminated oligo to the 5'end of the RNA

NH2-5'...............................3'OH

this will prevent concatamerisation of the oligo and only 3' end ligation
of it.

3. PCR between this oligo sequence and the vector sequence (make the oligo
large enough so that nested pcr can be performed at both ends).

4. Try out long PCR
5. clone products into T-vector



Martin
-- 

.....          Martin Leach                Email:leach at mbcrr.harvard.edu 
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323        
   / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329         
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