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fluorescent primer vs fluorescent terminator in sequencing


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#1 mushroom911

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Posted 02 September 2013 - 10:33 PM

Hi.  I got a question regarding the fluorescent primer and fluorescent terminator in sequencing. I wonder what are the advantages and disadvantages of both methods in sequencing.unsure.png

 

Thanks!!!!!smile.png



#2 mdfenko

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Posted 03 September 2013 - 07:07 AM

with fluorescent primers you have to run 4 separate reactions (one for each terminator) and 4 separation lanes (or capillaries, depending on your sequencing apparatus) because all terminated chains will have the same fluor.

 

with fluorescent terminators, each specific terminator will have a specific fluor and the entire sequencing reaction can be run in one tube and analyzed in one lane (or capillary). note that different fluors will have different charges and that will have to be taken into consideration when calculating the resulting sequence from the raw data.


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#3 mushroom911

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Posted 03 September 2013 - 05:29 PM

Thank a lot!!!smile.png

 

I got another question. rolleyes.gif I found this sentence in life technologies web site. 

"Dye primer chemistries generally produce more even signal intensities than dye terminator chemistries."

 

(under the sub-topic  5.What is Dye primer cycle sequencing?)

http://www.appliedbi...encing/faq.html

 

Is that mean fluorescent primer perform better than fluorescent terminator? But fluorescent terminator more cost, time and effort efficient since that you mention it can be run in just one tube?happy.png 



#4 mdfenko

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Posted 04 September 2013 - 06:05 AM

that statement may not be absolutely true.

 

primer labeling allowed for more even signal intensities when compared to internal labeling when using radioactive labels (internal labeling was generally done using alpha-35S-ctp). intensity was determined by the number of labeled nucleotides incorporated per molecule whereas you only had one label per molecule with primer labeling.

 

with terminator vs primer labeling you get only one label per molecule so intensity per molecule should, theoretically, be equal. abi seems to claim that "false stops" will affect intensity but false stops that are detected will give false sequence.

 

i noticed, from the faq link that you give, that abi uses 4 labels with primer and separates in one lane (or capillary). so you can disregard the 4 lane statement i made in the earlier post (still 4 reactions, though).


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#5 mushroom911

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Posted 05 September 2013 - 12:34 PM

Thank you! Your comment really help a lot! :)




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