Biomedical Engineering Reference
In-Depth Information
should share similar
T
m
values. Second, a given oligonucleotide
sequence should be unique to avoid multiple nonspecific binding
that may lead to incorrect assembly. The software must dynamically
choose the length of the oligonucleotides to ensure both the
specificity and the uniform
T
m
. The algorithm of FastPCR is able
to design oligonucleotides for long sequences containing repeats
and to minimize their potential nonspecific hybridization during 3′
end extension in PCR. For long sequence assembly, oligonucleotide
design starts from the 5′ end of a given sequence; the oligonucleotide
length is dynamically changed until a unique 3′ end has been found
and the
T
m
of the oligonucleotide has reached the
T
m
threshold. All
oligonucleotides are designed without gaps between them. The
other strand is used for the design of the overlapping
oligonucleotides using the same algorithm as above but with the
T
m
of the overlapping regions reaching the
T
m
-15 °C threshold.
The composition of the sequence at the 3′ terminus is important
because stability at the 3′ end of the double-stranded complexes
will improve the specificity of extension by the polymerase. To
reduce nonspecific polymerase extension (and ligation), the
algorithm chooses only unique sequences for the 3′ terminus.
Minimally, the last two nucleotides at the 3′ terminus must not be
complementary to any nonspecific targets. Other complementary
regions are less important for assembling multiple fragments by
PCR and ligation.
The input data can comprise either a single or many sequences.
Most of the parameters on the interface are self-explanatory. The
user is asked to provide the sequence and select oligonucleotide
design parameters. The user clicks on
Oligo options
on the
Oligos
Assembly
tab, and chooses the minimal oligonucleotide length
and
T
m
threshold, which by default are 40 nt and 60 °C, respec-
tively. The interface allows changing
T
m
calculation parameters.
The search process runs after pressing
F5
or from menu bar or
toolbox. The research result is presented as a list of oligonucle-
otides for both strands. On each strand, all oligonucleotides are
adjacent with no gap between neighboring primers. An oligonu-
cleotide will overlap two oligonucleotides from the complemen-
tary strand. The algorithm pays attention to avoid nonspecific
oligonucleotide hybridization to repeated regions. Where it is not
possible to design primers outside of repeated sequences, it is like-
wise difficult to find short specific oligonucleotides. The solution
to this problem is to divide the sequence into short segments,
design a set of oligonucleotides for each segment independently,
and then combine all these segments in the second PCR for final
amplification.
5.13 Polymerase
Extension PCR for
Fragment Assembly
Sequence-independent cloning, including ligation-independent
cloning, requires generation of complementary single-stranded
overhangs in both the vector and insertion fragments. Similarly,
