The degenerated primers were designed based on NGTVKV (Table 1, araN1, araN2, and araN3), and the occurrence of degeneration at 3rd base from 3′ terminus was a key point for obtaining specific amplifications (Fig. 2B-b).
When this position was not degenerated, the primer with a matched 3rd base (araN1) yielded the fragment of only one size (Fig. 2B-b, lane 4), and the primer with a mismatched 3rd base (araN2) yielded kinds of non-target fragments (Fig. 2B-b, lane 5); and when the primer containing both matched and mismatched 3rd bases (araN3), the PCR products showed all the contaminants (Fig. 2B-b, lane 6).
The difference in the specificity of PCR amplifications by the generated primers for the arabinosidase gene showed that araN3, a mixture of complementary and non-complementary 3rd base from the 3′-end, would change PCR format rather than produce the mixed fragments from araN1 and araN2 (Fig. 2B-b, lanes 4 6).
In order to compare the specificity of amplification caused by the degeneration at the 3rd base from 3′-end, three oligonucleotides were synthesized as primers annealing to the complementary sequence of the gene deduced from amino acid sequence NGTVKV, which were designated as araN1, araN2, and araN3 (Table 1).
It uses two cameras, suspended over the 1st and 3rd base lines, to follow the 60-foot, 6-inch (roughly 18-meter) flight of a pitched baseball.
For example, the 6th base of the FluA reverse primer was changed from "G" to "A", the 3rd base of the H7 reverse primer was altered from "C" to "T", and the 2nd and 23rd bases of the N9 forward primer and the 19th base of the N9 reverse primer were altered from "T" to "G", "T" to "G", and "A" to "G", respectively, relative to the novel H7N9 sequences.
