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dideoxy chain-termination method for sequencing DNA

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dideoxy chain-termination method for sequencing DNA

Postby thewax » Sun Dec 14, 2008 6:26 am

Three questions on dideoxy Chain-Termination Method for Sequencing DNA:
(1) - In DNA sequencing, how come scientists just don't tag regular deoxyribonucleotides with a specific fluorescent molecule (so that each type of nucleotide get a different color - one for A, one for G, one for T, and one for C) so that the DNA polymerase can build the complementary strand in just one try (so that you actually conserve nucleotides and materials). Then you would use a fluorescence detector to "sense the color of each fluorescent tag" (pg. 397) - the same procedure of identifying the tags as the dideoxy Cain-Termination Method for sequencing DNA. Wouldn't that also work?????
(2) - How to scientists synthesize the ddC, ddG, ddA, or ddT (I mean how do they take out the O or OH group from the 3' carbon)?????
(3) - In the seventh edition book Biology by Campbell and Reece pg. 397 figure 20.12 step #2, the figure shows strands of increasing length with the ddC, ddG, ddA, or ddT at the top of each, and where the dd_ were on the DNA strand right that is replaced with the correct deoxyribonucleotide and then the dd_ is on top of that. How does this process of Chain-Termination Method for Sequencing DNA ensure that the strands will be of increasing length ( I mean, if the DNA polymerase randomly does not pick the dd_ until much later, then how is it assured that the next strand will have not - randomly - pick the dd_ until much later)? of increasing length such that the next strand has the correct nucleotide and then the nucleotide immediately on top of that has the dd_? If the DNA polymerase did not pick the dd_ until much later (according to page. 397, step #2 - "Synthesis of each new arts at teh 3' end of the primer and continues until a dideoxyribonucleotide is inserted, at random, instead of the normal equivalent deoxyribonucleotide) and Chain-Termination Method for Sequencing DNA follows the figure shown, then I would have to conclude that the next strand will also not pick the dd_ until much later +1 nucleotide. And so on and so forth. So how are you ever going to figure out the DNA nucleotide sequence that follows directly after the primer?
Last edited by thewax on Sun Dec 14, 2008 7:22 pm, edited 1 time in total.
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Postby MrMistery » Sun Dec 14, 2008 3:42 pm

Could you please restate where exactly in this question you are having trouble. You're text is very disorganized and hard to read.
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Re: dideoxy chain-termination method for sequencing DNA

Postby domwood » Sun Dec 14, 2008 5:55 pm

To answer each point..
1. The distance between each nucleotide is smaller than any of our current mangnification equipment can resolve. You would not be able to see the different colors. Although there has been recent research into putting DNA sequences individually into nanotubes and measuring the chemical 'flash' when a nucleotide binds, but its still being developed.
2. This one I'm not sure of, not being a chemist, but it's probably not that difficult to synthesise these nucleotides chemically.
3. You might be misunderstanding the method here, the strands are of increasing length, but the strands haven't been produced sequentially. There will be millions of copies made, each one will end randomly, so for a 10 nucleotide sequence say you might get 3,2,8,1,2,9,2,3... But eventually you will get one of every length tagged with the appropriate fluorescent ddN. Obviously there are limits as the sequence gets bigger, which is why sequences are usually shotgunned and each fragment sequenced.
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Re:

Postby thewax » Sun Dec 14, 2008 7:24 pm

MrMistery wrote:Could you please restate where exactly in this question you are having trouble. You're text is very disorganized and hard to read.
The best I could do was to give each question their own line.

Thanks domwood for replying! :D
However, you were unsure of a couple of points... would anyone like to answer the three questions again to reaffirm domwood's answers???????
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Re: dideoxy chain-termination method for sequencing DNA

Postby thewax » Mon Dec 15, 2008 1:53 am

I have another question with domwood's answer (I'm not saying that it's wrong - all I'm saying is that I don't get it)...
for your answer to 1, to the best of my knowledge, the current mehtod of dideoxy chain-termination method for sequencing DNA does use a fluorescence detector (with a laser) to "sense the color of each fluorescent tag as the strands come through. Strands differing by as little as one nucleotide in length can be distinguished." (Campbell and Reece Biology 7th edition, page 397)
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Postby MrMistery » Mon Dec 15, 2008 4:56 am

thewax,

That is a legitimate question. You need to understand that in the chain termination method, after doing the amplification in the presence of ddNTPs you separate the molecules by size by electrophoresis. The electrophoresis is sensible enough to separate molecule that differ in only one base pair. However, what the laser detector sees at each step is not the individual tagged dideoxynucleotide, but actually the whole DNA molecule. In other words, you only see the tagged nucleotide because it is the only one tagged, but you don't see the actual nucleotide. To explain it better, imagine you had a satellite picture of a yacht in the middle of the ocean. If all the people on the yacht had a flashlight in their hand and pointed it up(the equivalent of DNA molecules that have the same length and the same tagged nucleotide at their 3' end) you might see the boat from space. However, if everyone had a differently colored bulb on their flashlight, would you be able to notice the individual colors from space? Of course not, you would just see an evenly colored blob. In the hypothetical situation presented to you in question 1, that is exactly the situation you are put before. What the question is asking is basically putting a DNA molecule under some hypothetical microscope and actually looking at it. However cool that may sound, it's not nearly possible.

If you would like to know more about a powerful method of sequencing DNA using real-time detection of incorporated nucleotides, I suggest you look into pyrosequencing. Keep in mind though that pyrosequencing does not have anything in common with chain termination sequencing, it is an entirely different approach.

Best,
Andrei
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Re: dideoxy chain-termination method for sequencing DNA

Postby thewax » Tue Dec 16, 2008 2:16 am

thanks MrMistery! :D

Just one question: then how does the laser and detector in the current dideoxy chain-termination method for sequencing DNA detect whether the dideoxy nucleotide is actually adenine, thymine, cytosine, or guanine if the laser can't detect the color of the "flashlight" (in the hypothetical situation)?
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Postby MrMistery » Tue Dec 16, 2008 3:26 am

the four nucleotides are tagged with florochromes of different colors(adenine is red, citosine is red etc). The laser can read the colors
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Re: dideoxy chain-termination method for sequencing DNA

Postby thewax » Wed Dec 17, 2008 5:33 am

the four nucleotides are tagged with florochromes of different colors(adenine is red, citosine is red etc). The laser can read the colors


Contradiction: cytosine and adenine are both red and yet their are of different colors????
Wait... if the laser can read the colors, then it must read the color off the last nucleotide (as if it showed up as a light on a boat, then if it were close enough to discern the color from the four possible colors, then it would theoretically be close enough to see the color of the different nucleotides - here I am referring to the new "method"). Then if it reads the color off the last nucleotides, then it would be close enough to read the color off the other nucleotides, which brings us back to the original question.

(1) - In DNA sequencing, how come scientists just don't tag regular deoxyribonucleotides with a specific fluorescent molecule (so that each type of nucleotide get a different color - one for A, one for G, one for T, and one for C) so that the DNA polymerase can build the complementary strand in just one try (so that you actually conserve nucleotides and materials). Then you would use a fluorescence detector to "sense the color of each fluorescent tag" (pg. 397) - the same procedure of identifying the tags as the dideoxy Cain-Termination Method for sequencing DNA. Wouldn't that also work?????
(2) - How to scientists synthesize the ddC, ddG, ddA, or ddT (I mean how do they take out the O or OH group from the 3' carbon)?????
(3) - In the seventh edition book Biology by Campbell and Reece pg. 397 figure 20.12 step #2, the figure shows strands of increasing length with the ddC, ddG, ddA, or ddT at the top of each, and where the dd_ were on the DNA strand right that is replaced with the correct deoxyribonucleotide and then the dd_ is on top of that. How does this process of Chain-Termination Method for Sequencing DNA ensure that the strands will be of increasing length ( I mean, if the DNA polymerase randomly does not pick the dd_ until much later, then how is it assured that the next strand will have not - randomly - pick the dd_ until much later)? of increasing length such that the next strand has the correct nucleotide and then the nucleotide immediately on top of that has the dd_? If the DNA polymerase did not pick the dd_ until much later (according to page. 397, step #2 - "Synthesis of each new arts at teh 3' end of the primer and continues until a dideoxyribonucleotide is inserted, at random, instead of the normal equivalent deoxyribonucleotide) and Chain-Termination Method for Sequencing DNA follows the figure shown, then I would have to conclude that the next strand will also not pick the dd_ until much later +1 nucleotide. And so on and so forth. So how are you ever going to figure out the DNA nucleotide sequence that follows directly after the primer?


Oh yeah, I also quoted the first and second questions because it seems that no one has yet to affirm domwood's answers.
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Re: dideoxy chain-termination method for sequencing DNA

Postby thewax » Tue Dec 23, 2008 1:22 am

can anyone help?
does anyone know the answer?
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