Biomedical Engineering Reference
In-Depth Information
multiple fragments can be joined or concatenated in an ordered
manner using overlapping primers in PCR. Annealing of the
complementary regions between different targets in the primer
overlaps allows the polymerase to synthesize a contiguous fragment
containing the target sequences during thermal cycling, a process
efficiency depends on the
T
m
and on the length and uniqueness of
the overlap. To achieve this, the program designs compatible
forward and reverse primers at the ends of each fragment, and then
extends the 5′ end of primers using sequences from the primers of
the fragment that will be adjacent in the final product. The input
sequence can be made of either a single or many sequences. The
user needs to pay special attention to the preparation of the given
sequences for assembly.
Users can specify the locations for both forward and reverse
primers design using “
[]
” to bracket the region. The bracketed
sequences will be used by the program for designing the overlap-
ping primers. The program selects the overlapping area so that the
primers from overlapping fragments are similar in size with opti-
mal annealing temperatures. The program adds the required bases
so that the
T
m
of the overlap is similar to, or higher than, the
T
m
of
the initial primers. Primers are tested for dimer formation within
the appropriate primer pairs. The user chooses
Polymerase exten-
sion cloning
(
OE
-
PCR
) on the
PCR Primer Design
tab and
selects the limit for multiple-PCR-compatible combinations of
primer pairs (default is 100). After specifying sequence inputs and
PCR primer design options, the user can execute the search task.
Once the design of the primer sets is complete, the result will
appear in two text editors:
PCR primer design result
and
PCR
fragments assembling compatible pair primers
. The text editor
PCR primer design result
window displays the individual PCR
primer design data, including the primer list and the compatible
primer pairs for all sequences whose primers are found. The
PCR
fragments assembling compatible pair primers
text editor col-
lects the final search result and presents it as a list of sets of com-
patible primer pairs for individual fragment amplification and
assembly. Figure
7
shows a sample result visualization window.
Modelling the hybridization of primers to targeted annealing sites
is the only way to predict PCR products [
7
,
24
,
40
-
44
]. The last
10-12 bases at the 3′ end of primers are important for binding
stability; single mismatches can reduce PCR efficiency, the effect
increasing with proximity to the 3′ terminus. FastPCR allows
simultaneous testing of single primers or a set of primers designed
for multiplex PCR. It performs a fast, gapless alignment to test the
complementarity of the primers to the target sequences. For in
silico PCR, a quick alignment to detect primer locations on the
reference sequence is performed by analyses of both strands using
5.14
In Silico PCR
