DNA -> RNA .....

RPSCOTT RSCOTT at IRRI.CGNET.COM
Sun Sep 26 19:02:31 EST 1993


> From:	IN%"<@CGNET.COM:sjchmura at midway.uchicago.edu>"  "sjchmura"    26-SEP-1993 02:59
> 
> Is the only way to make RNA from cDNA (say for a reverse transcriptase control)
> to insert the cDNA into a vector with a RNA polymerase start site?
> 
> Is there such thing as de novo random priming from DNA similarly to how
> random hexamers + RT function?
> 
> Steven Chmura


Dear Steven,

It is the sigma subunit of RNA polymerase which directs RNA synthesis
from a promoter in the DNA template. If only this subunit is removed, the 
polymerase retains its polymerizing activity (but only shorter fragemts
are produced) in the presence of random primers. Preferred conditions for 
effective and uniform transcription are as follows:

	1. high random primer concentration
	2. low  levels of nucleotide triphospates
	3. use native DNA as template instead of ssDNA for longer
	   transcripts

I'm not sure however if sigma-less RNA polymerase is marketed somewhere.

You'll probably be interested to see the ff. references:

*Tabor S. DNA-dependent RNA polymerases. In Current Protocols in Molecular Biology

*Chamberlin (1982) Bacterial DNA-dependent RNA polymerases. In The Enzymes
vol 15B.

Good day. :-)


R. Scott
Genetics Laboratory
Entomology & Plant Pathology Division
International Rice Research Institute
POB 933, 1099 Manila, Philippines
e-mail: rscott at IRRI.cgnet.com






 



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