An RNA question !!!!***

Shahram Mori smori at nmsu.edu
Tue May 10 00:43:55 EST 1994


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.

			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?

Thanks a milion for your input. Suggestions are welcome.
Cheers.
Shahram Mori
Program in Molecular Biology
Dept. of Chemistry Box 3C
NMSU   Las Cruces NM
88003


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