Primer design
Primer pairs were automatically designed using
PRIMER 0.5 (http://www-genome.wi.mit.edu/ftp/pub/
software/primer.0.5), with parameters essentially as described
by Chen.5 Primers were 18–34 nucleotides long,
devoid of secondary structure or consecutive tracts of a
single nucleotide, a GC content around 50% (Tm approximately
60◦C) and preferably G- or C-rich at the 3 end.
Sequences that provided no useful primer pairs were eliminated
from further consideration.