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question about bigdye sequencing reaction & capillary electrophoresis


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3 replies to this topic

#1 claritylight

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Posted 05 February 2010 - 05:06 AM

This question is directed for the ABI 3730 sequencers:

Capillary electrophoresis is based on separating the DNA fragments by size in the machine, and since these fragments are of different length, the smaller fragments will move faster than the larger fragments during capillary electrophoresis. But if the sequencer is detecting the base right before the ddNTP, how does it produce the final sequence so accurately if the fragments are chain terminated at random in the sequencing reaction? Wouldn't the random chain termination in the sequencing reaction cause a problem for the sequence to be read correctly on the sequencer? If the chain termination is random, then how does the DNA sequence get read correctly?

#2 Chakchel

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Posted 05 February 2010 - 06:04 AM

Hi,
just let's go it through step by step...
you use one primer for your sequencing PCR reaction. This should bind to only one area... :(
Here the strand starts.
All fragments end with a fluorescence-tagged ddNTP. Only the last one is a ddNTP, because the strands can't be elongated anymore when there is no 3'-OH. And only the fluorescence is detected, not any nucleoutides before you ddNTP...
Thus, all fragments that have the same size, also have the same ddNTP and the same tag.

You only get mixed signals when your primer binds at other regions, too, when your PCR reaction is not working properly and the wrong ddNTP is binding to the DNA, or when you have mixtures of templates (means again that your primer could bind to several regions).

Greetings,
Chakchel

#3 mdfenko

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Posted 05 February 2010 - 07:46 AM

in addition to what chakchel wrote:

If the chain termination is random, then how does the DNA sequence get read correctly?

there are a large number of random chains produced so that you will find all of the chains with 1 base (size) difference in the range of detection of the instrument.

this applies to all sanger sequencing methodologies.

Edited by mdfenko, 05 February 2010 - 07:48 AM.

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#4 Mazhar Hussain

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Posted 08 February 2010 - 01:21 AM

My Dear!
Let i try to explain...
There are billions of DNA molecules.... DNA polymerase incorporating dNTPs to it elongating the length of molecule.... Another thing ddNT is also present which competes for DNA polymerase to be inserted.... Once dideoxy is inserted DNA will not be elongated.... If billions of time same event is going to happen again and again there is probability that dideoxy will be inserted at each base position....

On the other hand optimization of PCR for sequencing is also important... The ratio of dNTPs and dideoxy bases is optimized by hit and trial method for best results....

I hope U get it....
Mazhar Hussain
Microbiology & Molecular Genetics
Website: Microbiology On-Line
FaceBook Group: Microbiology On-Line




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