RNA Sequencing Help!!!
David F. Spencer
dspencer at is.dal.ca
Wed Dec 24 11:05:46 EST 1997
In article <3496FC58.323A at zsu.edu.cn>, ls10 at zsu.edu.cn wrote:
> I want to sequence a 600bp mRNA. Is there any commercial kit for direct
> RNA sequencing? Should I have to retrotranscribe it into cDNA first? Any
> practical protocol and suggestion is warmly welcomed.
There are protocols for direct sequencing of RNA although few people today
seem to know that they even exist. Using reverse transciptase (RT) is
_not_ direct sequencing because the process ultimately involves DNA, the
product of RT.
In the late 70's several people in Walter Gilbert's lab did most of the
original work developing true "modern" RNA sequencing protocols, this of
course being just after the landmark Maxam and Gilbert paper on chemical
sequencing of DNA. Prior to that RNA sequencing was an extremely arduous
task involving T1 ribonuclease oligo fingerprinting (cataloguing) with RNA
that was labelled by in vivo P32 incorporation.
A paper by Donis-Keller, Maxam and Gilbert (Nucleic Acids Research
4:2527-2538, 1977) and an additional paper by Simoncsits et al. (Nature
269:833-836, 1977) described a method using limited ribonuclease digestion
of 5-prime labelled RNA (although it also works for 3-prime labelled) to
determine RNA sequence. Later Peattie (PNAS 76:1760-1764, 1979) described
chemical degradative methods (similar to the DNA protocols) that could be
used on 3-prime labelled RNA.
These direct methods can only read about 200 bases from either end so to
get sequence for longer RNA's additional methods must be used, today
usually involving c-DNA, cloning, etc. An obvious advantage of real
sequencing of the 3-prime ends of RNA is that an exact primer can be
designed for subsequent RT, c-DNA use, although most people now would tail
the 3'-end and prime with an appropriate oligo. The most important
remaining use for enzymatic and chemical sequencing of RNAs is with
structural RNAs (rRNAs, tRNAs), for determining the real ends of the mature
RNA and for studying modified nucleotides within such RNAs.
Pharmacia (now Amersham-Pharmacia Biotech) has a kit for chemical
sequencing RNA and other companies have in the past offered enzymatic kits
but I would suspect that you won't want to use this type of protocol. Even
RT sequencing can't read much beyond 200 bases from the primer so you will
have to generate c-DNA and do cloning if you want the sequence for the
entire 600 bases. You could of course alternatively do primer walking while
still employing RT on the RNA but the quality of sequence data for RT is
never as good as from DNA clones (as a consequence of using reverse
transcriptase and because the template is RNA) so that doesn't seem the
most practical choice.
David F. Spencer, PhD
Dept. Of Biochemistry
Halifax, Nova Scotia
dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca
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