Using Web Primer: Sequencing
Location (with respect to Gene chosen)
The segment of DNA that is analyzed to find sequencing primers is
determined by the values entered in the Distance boxes as well as the
gene entered. If the user wanted to sequence YAR024C and
approximately 1000 basepairs on either side of the gene then 1000
would go in each of the boxes.
The segment of DNA chosen is then chopped up into pieces. The length
of these pieces is represented by the spacing variable. If the user
entered 400, these pieces would be 400 basepairs in length. One
primer will be chosen from each of these regions.
The user is allowed to specify how big a region (the "length of DNA in
which to search for valid primers" field) in which to accept a
primer. If the user used the default 35 basepairs, then the FIRST 35
basepairs of each of the pieces of DNA created by the distance between
algorithm is analyzed for valid primers.
The lengths of valid primers are user definable and can be set by the
various "length" fields.
Location (with respect to DNA entered)
The segment of DNA entered is first chopped up into pieces. The length
of these pieces is represented by the spacing variable. If the user
entered 400, these pieces would be 400 basepairs in length. One
primer will be chosen from each of these regions.
The user is allowed to specify how big a region (the "length of DNA in
which to search for valid primers" field) in which to accept a
primer. If the user used the default 35 basepairs, then the FIRST 35
basepairs of each of the pieces of DNA created by the distance between
algorithm is analyzed for valid primers.
The lengths of valid primers are user definable and can be set by the
various "length" fields.
Primer composition
Primers that contain a skewed AT/GC ratio can fail to give high
specificity, or yield primers that are in other ways not well
behaved. The user is allowed to enter minimum, optimal, and maximum
values for the percentage of basepairs which are either G or C.
Primer Annealing
Primers also tend to dimerize and anneal to themselves. One method
for accounting for this problem was developed by Hillier and Green, PCR
Method. Applic., 1; 124-8, 1991. Maximum values annealing between
primers are able to be set by the user.
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