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dideoxy chain-termination method for sequencing DNA - (Dec/13/2008 )

I was wondering about 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?


I can see how you might have viewed this as a homework question...
However, it is not a homework question...
I hardly ever see questions worded like this, anyways...


There isn't just one strand of DNA in a PCR, there are millions - calculate if you will, how many copies you get if you take a single item and copy it so that you then have two, then copy those so that you have 4 etc, out to 30 cycles.

Another thing... have you heard of stochasticity?

Also, these might not be homework questions, but they sure seem like it, especially as a lot of places are having exams about now.


1) Are you answering another topic, because this topic is not about PCR...
Thanks for trying though... biggrin.gif
2) What is stochasticity? (I can't really find it online)


Actually, sequencing is all about PCR!

Stochasticity is the randomness in a system, in your question, stochasticity says that the addition of a ddNTP to a strand will be random, and as there are lots and lots of strands...


Have you gone to the other font of all knowledge, www.wikipedia? You could look up 'DNA sequencing', 'dideoxy sequencing' or 'Sanger sequencing'. You could go to any of the sequencer instrument manufacturers' websites and see what information they give about the reactions and processes. You might have a hard time getting useful information from a textbook.