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Dideoxy sequencing

Dideoxy sequencing

The most popular method of dna sequence determination (c.f. Maxam gilbert sequencing). Starting with single stranded template dna, a short complementary primer is annealed and extended by a dna polymerase. The reaction is split into 4 tubes (called a, c, g or T) each containing a low concentration of the indicated dideoxy nucleotide, in addition to the normal deoxynucleotides. Dideoxynucleotides, once incorporated, block further chain extension and so each tube accumulates a mixture of chains of lengths determined by the template sequence. The 4 reactions are denatured and run out on an acrylamide sequencing gel in neighbouring lanes and the sequence read up the gel according to the order of the bands.


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PCR

look for dideoxy sequencing

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by JackBean
Tue Jan 03, 2012 11:09 am
 
Forum: Genetics
Topic: PCR
Replies: 2
Views: 1544

Re: Dideoxy DNA sequencing?

Okay there's something I'm not understanding here and I'll probably feel really stupid in a minute. I thought the dideoxy nucleotides were tagged with different colours you could see under a microscope- so then I thought, why not tag the regular nucleotides and look at the colour ...

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by maccha
Thu Mar 04, 2010 1:27 am
 
Forum: Molecular Biology
Topic: Dideoxy DNA sequencing?
Replies: 6
Views: 1955

Re: Dideoxy DNA sequencing?

... each color fluor present, how would you know where in the sequence they are in along the DNA molecule? Terminating a growing chain with a single dideoxy allows you to run the mixture of different chain lengths out on a gel, sorting by size, with a different pure color at the end of each sized ...

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by jonmoulton
Wed Mar 03, 2010 12:31 am
 
Forum: Molecular Biology
Topic: Dideoxy DNA sequencing?
Replies: 6
Views: 1955

Re: Dideoxy DNA sequencing?

Couldn't you just look at the different fluorescent colours and see it from there?

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by maccha
Wed Mar 03, 2010 12:04 am
 
Forum: Molecular Biology
Topic: Dideoxy DNA sequencing?
Replies: 6
Views: 1955

Dideoxy DNA sequencing?

So I understand that fluorescent markers are attached to each of the dideoxy nucleotides and then the DNA fragments are aligned and read to determine their sequence.. why not attach fluorescent markers to the regular nucleotides and then be able to read the ...

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by maccha
Mon Mar 01, 2010 4:23 am
 
Forum: Molecular Biology
Topic: Dideoxy DNA sequencing?
Replies: 6
Views: 1955
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