Help Primer Extension

[Jayant Aphale]

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

>Hello,
>        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.

>                                                Jorge
>                                                University of London

There are 4 types of problems that can occur with primer extension.

1)  RNAase contamination

Solution:

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 
detection.

Solution:

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 
extension.

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 
was predominant.

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.
Glenn Soltes
Cangene Corp