i have two files from the DNA sequencer one for the foreword strand and the second for the reverse strand how the device separate them and does it can recognize the direction of the primer
As Stephane Roche mentioned, a sequencing reaction is different from a standard PCR reaction in two main ways
1) Each sequencing PCR reaction only has ONE primer present
2) The sequencing PCR reaction has dideoxy nucleotides which terminate the reaction.
If you sequence both forward and reverse, you will need TWO separate reactions. One with the forward primer, the other with a reverse primer. When the primer binds, you will extension by the polymerase but then at one point in time a dideoxy nucleotide will incorporate and top the reaction. This dideoxy also has an attached "color" - fluorophore.
During the reaction cycles, you will by chance have each position a terminating base, which are then separated by capillary electrophoresis.
Without getting into the details (There are lots of Sanger sequencing videos on you tube as well which may help), you will then indeed receive two files, which you can then align if there is over lap (depending on the distance between the two primers) You will need to know which was your forward primer reaction and your reverse primer reaction, so make sure you label them well.
A good sequencing run will usually give you from 600-800 good quality calls. Hence if your forward and reverse are 1000 bases apart, you should get a nice overlap.
Hope this helps.
They do two sequencing reaction, one with your forward primer and one with the reverse. No need to know the direction of each primer, both give a sequence from 5' to 3' in sens (for fowward) and antisens (reverse), if you have design your primer like that.
I agree with stéphane, and after, when you will have you sequences, you can blast them.
As Stephane Roche mentioned, a sequencing reaction is different from a standard PCR reaction in two main ways
1) Each sequencing PCR reaction only has ONE primer present
2) The sequencing PCR reaction has dideoxy nucleotides which terminate the reaction.
If you sequence both forward and reverse, you will need TWO separate reactions. One with the forward primer, the other with a reverse primer. When the primer binds, you will extension by the polymerase but then at one point in time a dideoxy nucleotide will incorporate and top the reaction. This dideoxy also has an attached "color" - fluorophore.
During the reaction cycles, you will by chance have each position a terminating base, which are then separated by capillary electrophoresis.
Without getting into the details (There are lots of Sanger sequencing videos on you tube as well which may help), you will then indeed receive two files, which you can then align if there is over lap (depending on the distance between the two primers) You will need to know which was your forward primer reaction and your reverse primer reaction, so make sure you label them well.
A good sequencing run will usually give you from 600-800 good quality calls. Hence if your forward and reverse are 1000 bases apart, you should get a nice overlap.
Hope this helps.
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