DNA sequencing.

[ecoli]

Does anybody have any good scientific/technical papers that explain this? I don't need actual papers, just references or links. I doing a sequencing reaction in my lab, but doing the assay and understanding what's going on are two different things. I'm trying to get a better grasp on what I'm doing. Thanks

[rakuenso]

are you using the dideoxy method or shotgun method? and what kind of sequences are we talking about? Genomic? Plasmid? or etc...

[ecoli]

dideoxy method, sequencing a plasmid. Sorry about the lack of detail.

[rakuenso]

if its just simple dideoxy kimball has a nice explanation on it:

 

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html

[ecoli]

thanks rakuenso, thats a good paper. Any others? Do they get more technical, perhaps?

[rakuenso]

http://www.nottingham.ac.uk/~mbzspd/methods/DNA_Sequencing.html

 

Here's one that lists reagents and buffers

[donkey]

are you using the dideoxy method or shotgun method?

I'm a bit confused, couldn't (wouldn't?) the shotgun method use dideoxy sequencing (i.e. it's not either or). My vague understanding is that shotgun sequencing is essentially a method used for assembling sequences together in order to reconstruct sequences of large stretches of DNA (such as a genome) whereas dideoxy (Sanger) sequecing tells us which actual base pair molecules appear in order.

[Yggdrasil]

Yes, the shotgun method is more of a method of assembling sequencs obtained from dideoxy sequencing into a full genome.

 

An alternative to dideoxy sequencing is chemical sequencing which uses chemicals which degrade specific base pairs. First you label the 5' end of DNA with a ³²P, then you would incubate the DNA with a low concentration of these chemicals for a specified time so that on average you have one clevage per DNA molecule. Electrophoresis of the fragments and visualization by autoradiography allows one to determne the sequence.

 

Chemical sequencing methods are not widely used anymore because it's much easier to automate dideoxy sequencing. Also, using fluorescently tagged ddNTPs is preferable to using the radioisotopes and hazardous chemicals required in chemical methods.

[BeYeu05]

I Scholar Googled "DNA Sequencing" and landed upon the following page:

 

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html