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Old 12-11-2014, 06:54 AM   #1
quratulain
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Default sequencing protocol

can anyone please illustrate the whole protocol of sanger sequencing in simple way that involves purification of PCR reaction, bigdye PCR reaction, purification...or please recommend any site which can clear the concepts....why each step is being done and the purpose of different reagents....i am specially confused about the PCR reaction that includes bigdye that how this reaction do the amplification as no polymerase and other reagents are added?

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Old 12-15-2014, 08:28 AM   #2
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.i am specially confused about the PCR reaction that includes bigdye that how this reaction do the amplification as no polymerase and other reagents are added?
I'll take one of your questions. "Big Dye" does include a polymerase, buffer, dNTPs and dye-labelled ddNTPs.

Please note that reaction, although performed on a thermal cycler, is not a PCR reaction. The "chain reaction" of PCR occurs because the products of one cycle become templates in the next. PCR leads to an exponential increase in target DNA amounts. A normal sequencing reaction includes only a single oligonucleotide to prime polymerization. By cycling the reaction you can linearly increase the number of product molecules produced. But these product molecules are not templates for subsequent cycles.

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Old 12-15-2014, 09:14 AM   #3
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Default sequencing protocol

thanks alot... by saying "A normal sequencing reaction includes only a single oligonucleotide to prime polymerization", you mean , we use only one primer?

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Old 12-16-2014, 04:17 AM   #4
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yes, that's what he meant.
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Old 12-16-2014, 04:57 AM   #5
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then what is meant by forward and reverse primer? they are not two?
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Old 12-16-2014, 05:54 AM   #6
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then what is meant by forward and reverse primer? they are not two?
They are 2, but you only put them into 1 sequencing reaction at a time. So to get the forward and reverse sequence you need to do 2 reactions and run the products in different capillaries.

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Old 12-16-2014, 07:07 AM   #7
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okay, i got this part.
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Old 04-08-2015, 03:51 AM   #8
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after the sequencing reaction in thermal cycler, are the products double stranded and as due to addition of ddnTPs, the reaction stops, so one strand will have ddnTP at its 3 prime end while other strand does not have ddnTP at its 3 prime end but regular dnTP, i am right? and are these double stranded products denatured in the genetic analyzer before detection by laser?
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Old 04-08-2015, 06:02 AM   #9
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after the sequencing reaction in thermal cycler, are the products double stranded and as due to addition of ddnTPs, the reaction stops, so one strand will have ddnTP at its 3 prime end while other strand does not have ddnTP at its 3 prime end but regular dnTP, i am right? and are these double stranded products denatured in the genetic analyzer before detection by laser?
The product strand would be double stranded up to the position the ddnTP was incorporated and single-stranded past that position.

No, we no longer denature these double stranded products, mainly because of cycle-sequencing.
With cycle-sequencing each template strand produces many product strands. As many as 1 new product strand per thermal cycle. For this reason, the ratio of product to template strands is high. >10:1. So denaturation isn't necessary as there is already a large pool of single stranded products present in the final reaction.

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Old 04-09-2015, 06:37 AM   #10
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but i don't understand that as products would be double stranded upto the position the ddnTP is incorporated and then single stranded, does all products would be not like that? denaturation isn't necessary as there is already a large pool of single stranded products present in the final reaction, from where only single stranded comes, they are not double stranded upto the position the ddnTP is incorporated and then single stranded?
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Old 04-09-2015, 09:07 AM   #11
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Yes, but at the beginning of each cycle the product strands from the previous cycle are melted from the template strands.

Details:
The sample starts out as normal double stranded DNA. The first step of cycle sequencing heats the sample up and the strands denature. Now the sample is single-stranded. Then the next step cools the sample so that the sequencing primers anneal to a specific place on the single stranded template. The temp is again adjusted so that the polymerases become active and extend the primers until they incorporate a ddNTP. That is the end of the cycle. Maximum possible yield: one product strand (of variable length) per template.

The second cycle begins as the first did, with a denaturation step. This melts all the product strands from all the template strands. Primer can anneal, etc.

Each cycle, the pool of product strands increases. So, at the end, even if a product strand anneals to each template, there will be plenty of extra product still left without template available to anneal to.

The truth is, in ancient times, 10+ years ago, many of us denatured samples just before loading them on the sequencer. But it was found not to be necessary, so these days most people don't bother.

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Old 04-09-2015, 04:46 PM   #12
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okay, i got this part.

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Old 04-10-2015, 10:02 PM   #13
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can i sequence 44 samples at a time in 3130 ABI genetic analyzer and how long will it take for their capillary electrophoresis?
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Old 04-11-2015, 06:36 AM   #14
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can i sequence 44 samples at a time in 3130 ABI genetic analyzer and how long will it take for their capillary electrophoresis?
I don't have a 3130 but I think they have either 8 or 16 capillary arrays. So it will be able to process either 8 or 16 samples per run. Typically ABI instruments will process runs in 2-4 hours, depending on the array length and maybe the polymer used. Also, the robotics will be able to do additional runs past the first until your plate is complete. Each run will consume additional polymer, though.

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Old 04-12-2015, 09:18 AM   #15
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what is the specific role of Hi-Di formamide?
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Old 04-13-2015, 07:45 AM   #16
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what is the specific role of Hi-Di formamide?
Formamide is just the solvent in which the reaction products are resuspended after "cleaning-up" the reaction. "Hi-Di" is said to be of high quality -- we were told it isn't oxidized like other sources of (cheap) formamide would be.

Back before cycle sequencing was common for generating reaction products, it was critical to denature the products from the template strand before subjecting them to electrophoresis. But after denaturation formamide might have been added to slow the renaturation of products back to template strands.

That said, the main reason to use it now would be because it generally produces much more even signal electrokinetic injections. That is, prior to electrophoresis the samples must be moved into the polymer-filled capillaries. So the tips of the array are dipped into the wells of a 96- or 384-well plate in which the samples have been resuspended. Then a brief burst of high voltage electrophoresis occurs that moves a portion of the reaction into each capillary. This process is called "electrokinetic injection". Then the tips are move over to the buffer tray and normal electrophoresis commences.

Only a tiny percentage of the reaction actually moves into the capillaries. If you use pure water (18.3 M-ohm/cm) to resuspend the samples instead of formamide, much (~5x) higher signal results. However, this results from molecules farther from the tips of the capillaries electrophoresing in than occurs with formamide solvation. As a result shorter products will preferentially be injected because they have lower mass and will move more quickly in the applied voltage difference. So signal of the longer products suffers at the expense of the shorter products.

Formamide, probably just by limiting the strength of the local field, greatly ameliorates this issue. I would say this is the main reason that formamide is used to solvate samples these days.

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Old 04-22-2015, 09:17 AM   #17
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please tell if any one knows that is it enough to exclude a gene on the basis of sequencing that gene in only one affected member of the autosomal recessive hearing loss family or do i need to do sequencing of the gene for all the affected members to exclude that particular gene in a family?
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Old 04-22-2015, 09:21 AM   #18
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please tell if any one knows that is it enough to exclude a gene on the basis of sequencing that gene in only one affected member of the autosomal recessive hearing loss family or do i need to do sequencing of the gene for all the affected members to exclude that particular gene in a family?
Please create a new thread for this. People are likely to miss this question buried in a thread titled "sequencing protocol".
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