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question on sequencing

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question on sequencing

Postby poloke » Mon Jun 18, 2012 8:46 pm

I am learning about sanger sequencing and I do not understand the following (from wiki): "Common challenges of DNA sequencing include poor quality in the first 15–40 bases of the sequence and deteriorating quality of sequencing traces after 700–900 bases"

How come the first bases are bad?

ANy ideas?
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Postby canalon » Tue Jun 19, 2012 2:51 am

The first fragments usually have large overlapping peaks because there are a lot of the short fragments. That means very strong (saturating and leeching light beyond its location) signal and wide, less defined band.
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Re: question on sequencing

Postby wbla3335 » Tue Jun 19, 2012 1:19 pm

poloke wrote:How come the first bases are bad?

Also, if you don't have a machine and incorporate radioactivity into your DNA, the short molecules will have weak signals.
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Re:

Postby poloke » Thu Jun 21, 2012 9:08 pm

canalon wrote:The first fragments usually have large overlapping peaks because there are a lot of the short fragments. That means very strong (saturating and leeching light beyond its location) signal and wide, less defined band.

So you that if the fragments where longer, the fluorescent label would not be such a big problem?
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Postby canalon » Fri Jun 22, 2012 1:52 am

The number of fragment of each size depends on probability. The shorter ones are the most common. There is one fluorophore per fragment. So the the very numerous short fragment are very bright, to the point that they can be hard to read correctly.
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Re:

Postby poloke » Mon Jun 25, 2012 11:39 am

canalon wrote:The number of fragment of each size depends on probability. The shorter ones are the most common. There is one fluorophore per fragment. So the the very numerous short fragment are very bright, to the point that they can be hard to read correctly.


Ah yes, I see what you mean!
Thanks a lot.
This is helpfull.
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