automated dna sequencing

Phillip SanMiguel pmiguel at bilbo.bio.purdue.edu
Thu Oct 3 18:19:20 EST 1996


In article <531dqt$h8q at newsbf02.news.aol.com>, apadmanabh at aol.com
(APadmanabh) wrote:

> Hi,
> 
> I'd like someone to answer the following questions:
> 1. How to TEMED, acrylamide and APS polymerize to form the gel?
Acrylamide is a monomer that in the presence of free radicals forms
a linear chain of connected monomers -- or linear polymer.  APS provides
the initial free radicals and TEMED is a catalyst.  The details of the
reaction I'm not sure of.  To make a gel you need a crosslinker such
as BIS-acrylamide that hooks together the linear chains into a 3D
structure of a density dependent on the percentage of acrylamide in it.

> 2. EDTA is a chelating agent.  What is a chelating agent?
A chelating agent binds metals.  (I think it's always metals -- maybe
some types of chelating agents could bind other types of atoms...)

> 3. Why is dna denatured before it is loaded?

To separate labelled product strands from the unlabelled template strand.
Separate, the difference between an N-mer and an N+1mer is easier to assay
on a gel.

> 4. Why is the dye terminator method considered more robust?

I've never done a dye terminator reaction but in principle it should 
label only successfully terminated products -- since the terminator is
the label.  Thus less noise.

> 5. When you call bases, during analysis, what exactly are you doing?
> 
Generally a computer program "calls" the bases, although a human could
(and must when manual sequencing).  Calling is identifying the
base at a certain position.
If I do call a base I'm looking a computer screen with a chromatogram on
it -- each base chromatogram a different color.  Where I see a peak I
"call" a base.  Or more than one base of the same type.
If I am reading a manual sequencing gel (which I thankfully no longer
need to do.) I look a sets of four sequencing reactions (1 with a "ddG"
terminator, 1 with a "ddA", 1 with a "ddT" terminator and 1 with a "ddC"
terminator) run in lanes next to each other.  I start at the bottom of the
gel (actually an autoradiogram of the gel) and push a straight-edge up.
For each descrete position on the gel as I go up there should be a band in
only one of the four reactions.  If that band is in the ddG terminated
reaction lane I call it a "G", etc.  
> Regards,
> 
> Arti



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