Biomedical Engineering Reference
In-Depth Information
which to add the extra bases and “NN” is a given sequence of
one to more bases. For example, “
-F5eCGACG -R5eTTTTTT
”,
means adding the sequence “
CGACG
” to forward primers and
“
TTTTTT
” to reverse primers, both at the 5′ ends.
5.6 Bisulphite-
Modified DNA
The “С ≫ T bisulphite conversion” option allows the design of
specific PCR primers for in silico bisulphite conversion for both
strands. Only cytosine not followed by guanidine (CpG
methylation) will be replaced by thymine:
5.7 Uniqueness
of Primers
Optionally, the user can synchronize the primer test for secondary,
nonspecific binding with a dataset of sequence names. The
program recognizes that a given sequence in the screening library
dataset (from loading the dataset file) is the same as the sequence
for which it is designing primers; this allows sequence selection to
be made even if the selection matches the screening sequence
perfectly. This allows the same dataset to be used for both primer
design and screening without having to make a new screening
database for each sequence. In other words, a dataset that contains
sequences A, B, C, and D can be used both for choosing primers
and for checking primer specificity. Alternatively, the user can
input preexisting primers into a second “Additional sequence(s)
or pre-designed primers (probes) list” text editor. These primers
or probes will be checked for compatibility (inter-primer-dimer
formation) with newly designed primers. The number of
preexisting primers is not limited to one or two; it can be as many
as the user needs.
5.8 PCR Primer
Design
The PCR primer design algorithm generates a set of primers having
a high likelihood of success in virtually any amplification protocol.
All PCR primers designed by FastPCR can be used for PCR or
sequencing experiments. The program is able to generate either
long oligonucleotides or PCR primers for the amplification of
gene-specific DNA fragments of user-defined length. FastPCR
provides a flexible approach to designing primers for many
applications and for both linear and circular sequences. It will
check if either primers or probes have secondary binding sites in
the input sequences that may give rise to additional PCR products.
The selection of the optimal target region for the design of long
oligonucleotides is performed in the same way as for PCR primers.
The basic parameters in primer design are also used to measure the
oligonucleotide quality and to evaluate the thermodynamic stability
of the 3′ and 5′ terminal bases.
