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Pyrosequencing questions


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#1 One dollar lab

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Posted 10 June 2009 - 11:26 PM

My supervisor would like me to work out these questions in 1-2 days and nobody I know has pyrosequencing experience so any reply is appreciated!

1. For each sequencing reaction, Pyrosequencing Assay Design Software from Qiagen design a pair of PCR primer and a sequencing primer. Why the software design a sequencing primer? Why can't just use forward PCR primer for sequencing? What gonna happen if use PCR primer for sequencing?

2. For anyone who had experience in pyrosequencing, is there anything can go wrong? Any issue I need to be aware?

3. What optimization experients do I need to do before testing tissue samples?

Please tell me anything you know about any of the questions above! Thanks!

#2 Michaelro

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Posted 11 June 2009 - 11:43 AM

My supervisor would like me to work out these questions in 1-2 days and nobody I know has pyrosequencing experience so any reply is appreciated!

1. For each sequencing reaction, Pyrosequencing Assay Design Software from Qiagen design a pair of PCR primer and a sequencing primer. Why the software design a sequencing primer? Why can't just use forward PCR primer for sequencing? What gonna happen if use PCR primer for sequencing?

2. For anyone who had experience in pyrosequencing, is there anything can go wrong? Any issue I need to be aware?

3. What optimization experients do I need to do before testing tissue samples?

Please tell me anything you know about any of the questions above! Thanks!

Hi
I may give You the answer on topic #1
Since the length of the pyrosequencing run is short (~50bp), the software picks sequencing primer to be as close as possible to the region of interest on the amplicon
They don't like to perform the sequencing with PCR primers maybe because of possible contamination of the amplified DNA with primer dimers.
Anyway, I intend to use PCR primer in number of my pyrosequencing reactions and I'll see what happens
Good Luck

#3 One dollar lab

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Posted 14 June 2009 - 07:21 PM

Thanks a lot! That info is useful! I'll try a pyrosequencing after my experiment design is approved by my supervisor.

My supervisor would like me to work out these questions in 1-2 days and nobody I know has pyrosequencing experience so any reply is appreciated!

1. For each sequencing reaction, Pyrosequencing Assay Design Software from Qiagen design a pair of PCR primer and a sequencing primer. Why the software design a sequencing primer? Why can't just use forward PCR primer for sequencing? What gonna happen if use PCR primer for sequencing?

2. For anyone who had experience in pyrosequencing, is there anything can go wrong? Any issue I need to be aware?

3. What optimization experients do I need to do before testing tissue samples?

Please tell me anything you know about any of the questions above! Thanks!

Hi
I may give You the answer on topic #1
Since the length of the pyrosequencing run is short (~50bp), the software picks sequencing primer to be as close as possible to the region of interest on the amplicon
They don't like to perform the sequencing with PCR primers maybe because of possible contamination of the amplified DNA with primer dimers.
Anyway, I intend to use PCR primer in number of my pyrosequencing reactions and I'll see what happens
Good Luck






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