Universal DNA barcode regions of Substituent/ adulterant species (barcode seq. 1 as described in 188.8.131.52) were obtained by common primers (table 6). When using a blast search for this barcode seq. 1 with specifying the genus/ species name of the herbal plant, rarely it gives species specific primers. Although this method can be used to distinguish substituent/adulterant species from the original herbal materials, when it comes to species specific identification the best method of getting barcode seq. 2 is obtaining the best hit from the blast search of barcode seq. 1, which means two barcode sequences have higher similarity with fewer mismatches. Since the number of mismatches is less those mismatches are highly specific for the particular sequence. Primers were designed by assigning the mismatch at the 3′ end of the primer. This mismatch is limited to one or two base pairs. This type of primers is called “allele-specific primers”. Some issues have been identified with allele specific primers, as they have the specificity at the last base of 3′ end. Those issues were,
· When synthesizing primers, the full primer may not be produced.
· The specificity of primers lies at the final base. As the time progresses the primer bases tend to be degraded and the final base could be removed.
· Also due to improper handling during the experiments, these primers could be degraded.
To overcome those issues, primers have to be purified and then it has to be verified whether the primer contains its full length before admitting with the PCR. If the primer synthesized its length completely, then it could be assumed that above issues have been sorted out.
PCR product of allele specific primer pair depends on the type of the mismatch. Primer pairs with A-C, G-T could be amplified aspurine:pyrimidine (A-C, G-T) mismatch is much stabilize than purine:purine(G-G, A-A, G-A) and pyrimidine:pyrimidine(C-C , T-T, C-T) mismatches(Ralph et al., 2010).
According to the Primer-Blast results in section 184.108.40.206 except one primer pair all the others satisfying the above requirement of allele specific primers. There could be a solution for the primers that ended up with A-C, G-T to avoid the formation of PCR product.
220.127.116.11. Designed primers using internal variations
By considering dissimilar internal regions of DNA barcode sequence, obtained forward and reverse primers for selected plant species.