The Sanger Method

[jhirlo]

I believe you all know something about this method for sequencing dna, thing that confuses me is the first, or the first few nucleotides. How they sequence them, I guess you have to put some primers to turn on dna polymerase, if you do so you can’t sequence the beginning of the chain.
And how much and what kind of primers they put in ? If you want dinucleotide primer, and you don’t know seq of those two nucleotides, you’ll have to put in 16 different primers, I’m I Wright or not :smile: (probably not:)

[iansmith]

You migth want to try to look at the original paper by Sanger
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=4616099

You can also determine DNA sequence using the Maxam and Gilbert mehtods which do not require primer and could potentially allows you to design primers to be used in the Sanger method.
http://www.pnas.org/cgi/content/abstract/74/2/560

[jhirlo]

C’mon people :) you know this…
Sanger: one strain dna incubated with deoxynucleotides and fluorescently labeled dideoxy nucleotides (dd). You pass in primers and polymerase and then analyze, replicated chains, ended with dd nucleotide.
Again what about the start of sequence?

Q:
p.s. is there some compound of choice for covalent protein Cross-linking?

[jhirlo]

You migth want to try to look at the original paper by Sanger
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=4616099

You can also determine DNA sequence using the Maxam and Gilbert mehtods which do not require primer and could potentially allows you to design primers to be used in the Sanger method.
http://www.pnas.org/cgi/content/abstract/74/2/560
Ian, thanks again.
We must have wrote reply in same time.

I thought so too for using Maxam and Gilbert method.

p.s. I don’t have access to PubMed :(

[iansmith]

The paper are old so might to go to the library of institution to have access to the journal.

Sanger F, Donelson JE, Coulson AR, Kossel H, Fischer D.e. Determination of a nucleotide sequence in bacteriophage f1 DNA by primed synthesis with DNA polymerase. J Mol Biol. 1974 Dec 5;90(2):315-33.