Help Primer Extension
biotech at inforamp.net
Fri Aug 18 11:04:26 EST 1995
In article <Pine.SOL.3.91.950816133813.20207A-100000 at iron> "Mr. J. Membrillo-Hernandez" <jmembril at hgmp.mrc.ac.uk> writes:
>From: "Mr. J. Membrillo-Hernandez" <jmembril at hgmp.mrc.ac.uk>
>Subject: Help Primer Extension
>Date: Wed, 16 Aug 1995 13:40:21 +0100
> I have tried to identified the trascriptional start site for a
>gene in E. coli, however I have had problems doing it. I have follow the
>protocol in Maniatis et al. and did not work, could anybody help me to
>have a good protocol for primer extension?, thanks in advance.
> University of London
There are 4 types of problems that can occur with primer extension.
1) RNAase contamination
Careful "RNAase free" treatment of samples
DEPC treat solutions (except those with NH4)
2) Poor quality RNA or the mRNA levels for the gene are below the level of
Use a commercial kit such as Qiagen "RNAeasy" to isolate total RNA. It will
be pure, intact and you don't need tedious phenol extractions.
First perfect the primer extension technique with a gene that is well
expressed and has high mRNA levels. Use a well characterized gene expressed
from a high copy number plasmid that has already been mapped by primer
The gene you are interested in may have naturally low mRNA levels. Try
different growth conditions. Try to detect the mRNA using slot blot on RNA
which has been treated with RNAase free DNAse. The gene may also be
repressed or autoregulated. In this case a high copy number plasmid will not
help. Can you see a protein on a gel. Stable proteins may look well
expressed when in fact they have low mRNA levels.
3) Primer problems
Is the primer antisense to the mRNA?
Does the primer work well in a sequenceing reaction? If not, forget using it
for primer extension.
The primer should be designed so that the extended product is 40-150 bases
larger than the primer. Is you have no idea where the start site is you have
to make antisense primer sets (bottom strand) starting 40 bases downstream
from the translation start with another primer upstream every 50 bases. Use
these primers in a slot blot to roughly map the 5' end of the mRNA. It is a
good idea to do this anyway because there is often multiple promoters
separated by 100s of bases. One primer may miss transcription start sites
of mRNAs with short or long untranslated regions
Try primer extension with various primer annealing temperatures. The
calculated melting temperatures of RNA:DNA hybrids with short oligos is a
crude approximation at best.
Secondary structures are known to pause reverse transcriptase. I have seen
artifact bands associated with hairpins but the full length extended product
4) Technical Problems
It is easy to lose the pellet containing the extended product during the final
precipitations. Monitor waste ethanol for radioactivity to ensure you are not
losing counts. Wash with 80% ethanol instead of 70%
Do a short sequencing run to check if you can see a unextended labelled primer
band of the correct size. It should be be a very strong band.
I hope this helps you with your problem.
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