Non-Radioactive Sequencing (Manual)

[Wasun Chantratita]

On Wed, 30 Mar 1994, G K L Munro wrote:

> Does anyone have experience with manual sequencing of DNA using non-
> radioactive techniques?   In particular I would be interested in hearing
> from anyone using Promegas' Silver Sequence or Amershams kits.
> 
Dear Munro
	Silver DNA Sequencing (Promega) can be performed as a conventional
radioactive, Sanger DNA sequening method. After electrophoresis through 
polyacrlamide-Urea gel. The gel could then be stained with silver staining 
solution. The sharp bands will be appeared within 5min.
	Though the bands are sharp but quite weak (faint). Very often that 
you have to place stained gel attached to plate (gel side up) on a 
fluorescent light box and then place the EDF film on top of the gel. Turn 
the light on for 20 seconds. The film can be developed using the same 
procedure for development of autoradiographic film. Another advantage is 
you can use any kind of sequencing primers without labeling with any 
nonisotopic ligant.

	*Disadvantages*
	1) Since there is no amplification step, just only staning newly 
synthesized DNA in the gel with silver staining solution therefore you 
have perform DNA cycle sequencing to produce enough DNA. You have to use 
huge amount of DNA template (ug level) and primers.
	2) Background is extremely high. You have to carefully adjust 
time for silver staining and exposure with fluorescent light. a few 
seconds delay can cause high background. Everyone seems to know that 
sensitivity is depend on two factors, signal and noise ratio (background).



	Do you have any comment on this issue, Dr. Laura K. Moen?

Wasun
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My apologies for any error in my English and your correction would be 
appreciated.
Thank
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Wasun Chantratita, Ph.D.                        Phone:053-221122 Ext 5086,5068
Department of Clinical Microbiology             Fax:  053-221890 
Faculty of Associated Medical Sciences       Email:asmsi002 at cmu.chiangmai.ac.th
Chiang Mai University
Chiang Mai 50200
Thailand
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