An RNA question !!!!***

Olav Hungnes ohungnes at bioslave.uio.no
Tue May 10 10:02:45 EST 1994


Shahram Mori (smori at nmsu.edu) 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.

: 			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? 

I have no experience with RNA ligation, unfortunately. 
If the RNA ligase also joins dsDNAs, you must guard yourself against the 
false PCR template arising from self-ligation of the DNA:

    primer site
    ************                5'P***************same DNA
DNA **************5'P    +           ************* 
                                        primer site

One solution would be to cut with an enzyme which generates 
non-compatible ends, e.g. HgaI.

: 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?

Do you propose to use random oligos as PCR primers? For a given template, 
will not a limited set of suitable primer sequences be rapidly exhausted? 
Besides, one would think that the melting temperatures of the 6-mers are 
way too low for PCR. If you mean to use the 6-mers to make 
double-stranded cDNA this should not be necessary for the PCR; one 
complete DNA strand is sufficient.
Wouldn't it be better to ligate a non-phosphorylated oligo to the 5'P of 
the RNA first, and use the same oligo as PCR primer?

: 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


:   _/\_
:  _\  /_
:  \_  _/
:    ||

Good luck,
Olav
--
_______________________________________________________
Olav Hungnes                     ohungnes at embnet.uio.no
National Institute               Phone  (+47)22042200
of Public Health                 FAX    (+47)22353605
Oslo, NORWAY
_______________________________________________________



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