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BigDye Terminators


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Hello all,

 

I just started working in a genomics lab and we use Sanger sequencing. The sequencing protocol utilizes the BigDye 3.1 Terminator Kit and a BigDye XTerminator for clean-up. I did some online searching but to no avail couldn't really figure out what was going on during the BigDye Terminator reaction. Can anyone tell me what the primary agents are in the buffers and what they do e.g. Taq polymerase and MgCl2 or something of that sort? I'm just curious as to what is actually going on in these steps.. I am also a bit confused as to how chain termination works. Won't ddNTPs prevent DNA elongation?

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BigDye has regular dNTPs in it as well as the labelled ddNTPs at a ratio of about 100:1 or something like that. The PCR amplification product of a BDT reaction will therefore contain a mixture of your template of different sizes since the ddNTP's will incorporate randomly during the reaction, with the last bp of each chain being a fluorescently labelled ddNTP. The capillary on the sequencing platform will separate these based on sizes, with the smallest one coming out first. The machine will then detect the last bp of each chain that passes the laser and determine the identity of that base depending on the fluorescent label it has attached to it. Doing that allows it to compile the sequence of your template in the correct order, provided it isn't contaminated or botched in some other way. So yes, the ddNTP's will stop elongation, but that's kind of the point.

The BD mixture is proprietary and I doubt you'll find out exactly what's in it or in what amounts unless you have a very knowledgable FAS who is bad at keeping secrets. There will obviously be some kind of DNA polymerase in there, but it's likely that it will be some sort of specially modified Taq. Not sure about the buffer either, though you may be able to get some idea of what it is by finding the MSDS that goes with it.

 

Edit: I found this picture, which will hopefully make some more sense of it to you. the only part it doesn't explicitly mention is the fact that the polymer inside the capillary separates the various lengths of template by size, which is what allows us to determine the order in which the bases appear in (as I mentioned above). For example, in the example they show the top strand that terminates at position 1 would elute first and would be detected by the machine as being a G. The strand below that, which is one base pair larger and has the fluorescent ddNTP at the second position would then elute and the machine would recognise the terminating bp (i.e. the second bp) to be a G. This keeps on going until eventually you have a complete or near to complete picture of the sequence of your PCR product.

 

673px-Sanger-sequencing.svg.png

 

(source: http://en.wikipedia.org/wiki/Sanger_sequencing)

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