Biomedical Engineering Reference
In-Depth Information
Additionally the software allows this test to be performed
against a reference sequence or sequences (e.g., a BAC or YAC) or
one's own database. Primers that bind to more than one location
on given sequences will be rejected. Even though the test for non-
specific primer binding is performed as a default for all primers, the
user may cancel the operation. Identification of secondary binding
sites including mismatched hybridization is normally performed by
considering the similarity of the primer to targets along the entire
primer sequence. An implicit assumption is that stable hybridiza-
tion of a primer with the template is a prerequisite for priming by
DNA polymerase. FastPCR pays particular attention to the 3′ por-
tion of the primers and calculates the similarity of 3′ end to the
target (the length is chosen by user) to determine the stability of
any potential interactions.
The secondary nonspecific primer binding test is based on
repeat masking using a quick local alignment screen (which allows
one mismatch within a hash index of 12-mers) between the refer-
ence and input sequences.
5
Methods
Once the input files are selected or sequences copied and pasted to
the
General Sequence
(
s
) text editor, the FastPCR provides vari-
ous execution features. Figure
3
provides an example for primer
design from the user's perspective.
5.1 Execution of the
Selected Task
The user selects the ribbon having the task needed. The program
will only perform the selected task. The name of the selected
executive task is shown on the status bar by “Press F5.” The task is
executed by using key
F5
or by clicking the arrow on the toolbar
using the mouse. Once the executive task is completed, the result
is shown in the
Result report
text editor (e.g.,
see
Fig.
3
).
5.2 PCR Primer
Design Options
The “PCR Primer Design” Tab contains various execution
options for commonly selected types of PCR and for the most
important PCR parameters (Fig.
1
). The option panel of “PCR
Primers or Probe Design Options” is shown in Fig.
4
. Once the
user selects any attribute, the option attribute value field shows
the default attributes value, which can then be modified. “PCR
Primers or Probe Design Options” affects all sequences. PCR
primer design options can be customized for each sequence using
special commands at the header of the sequence (
http://
primerdigital.com/soft/pcr_help.html)
.
Typically, it is not
necessary to use these commands to manage typical PCR primer
design and these are applied to advanced tasks. Default global
parameters for primer design will be assigned by typing the help
command “
/?
” in text editor:
