FastPCR Software for PCR, In Silico PCR, and Oligonucleotide Assembly and Analysis - DNA Cloning and Assembly Methods - page 287

Fig. 6 An example of group-specific PCR results

FastPCR identifies all SSRs within each entry sequence and
designs compatible PCR primer pairs for each SSR locus. The
default PCR primer design parameters are that the primers must be
within 100 bases from either side of the identified SSR. Often the
sequences available around SSR loci are not suitable for designing
good primers; the user can increase or decrease the distance from
either side to find more efficient and compatible primer pairs. The
capabilities of FastPCR make it a complete bioinformatics tool for
the use of microsatellites as markers, from discovery through to
primer design. For example, the user can specify PCR primer
design to SSR loci within 200 bp around an SSR, with the com-
mand: “ -ssr/200. ” The software finds all SSR sites and then
will design PCR primers and compatible primer pairs indepen-
dently for each SSR locus.

The application to make long synthetic DNA molecules relies on
the in vitro assembly of a set of short oligonucleotides, either for
LCR [ 37 ] or for assembly PCR [ 38 ]. These oligonucleotides
should be adjacent on the same strand and overlap the
complementary oligonucleotides from the second strand. There
are two major parameters for designing oligonucleotides for gene
synthesis for LCR or assembly PCR. First, the oligonucleotides

5.12 Oligonucleotide
Design for In Vitro
Long Sequence
Synthesis