Dideoxy DNA sequencing?

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maccha
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Dideoxy DNA sequencing?

Post by maccha » Mon Mar 01, 2010 4:23 am

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 sequence straight from there?

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JackBean
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Post by JackBean » Mon Mar 01, 2010 9:28 am

how would you read it? You need to separate the strands by length and THAT tells you the order ;)
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Re: Dideoxy DNA sequencing?

Post by maccha » Wed Mar 03, 2010 12:04 am

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

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jonmoulton
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Re: Dideoxy DNA sequencing?

Post by jonmoulton » Wed Mar 03, 2010 12:31 am

How will you separate out the color signals if they are all mixed together? Even if you could sort them spectrally and learn the relative amount of 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 molecule. That way you simplify the signal (single color) and sort the molecules by size so you can read off the sequence as colors arranged along the gel lane.
Last edited by jonmoulton on Wed Mar 03, 2010 4:01 pm, edited 1 time in total.

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JackBean
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Post by JackBean » Wed Mar 03, 2010 3:18 pm

you had to extend the DNA strain and look for the colors under some really good microscope, where you would be able to distinguish only one molecule (which does not exist;)
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maccha
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Re: Dideoxy DNA sequencing?

Post by maccha » Thu Mar 04, 2010 1:27 am

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 sequence from there?

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JackBean
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Post by JackBean » Thu Mar 04, 2010 11:07 am

if you had many of them together, you can see them under microscope, but if you have only one molecule, you would need much higher resolution (like X-ray), otherwise we would not need to crystalize proteins :)
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