Normalization of samples and amplicons in multiplex PCR
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Optimised multiplex PCR amplification of DNA barcoding primers is essential for converting tedious Sanger based sequencing to robust and automated NGS based Sequencing. In addition, the normalisation of PCR product yield between samples and targets is required for successful conversion of Sanger sequencing to NGS. Spices and medicinal plants industry suffers from low cost substitutions and adulterations of herbs with closely related species. DNA barcoding offers accurate means to identify the plant species of interest as well as adulterants. Majority of DNA barcoding work is currently carried out by tedious Sanger sequencing of rbcL, matK, rpoB, trnH-psbA, and ITS gene markers. In this study, we have optimised a multiplex PCR method for simultaneous and uniform amplification for multiple plant DNA barcode targets in a single PCR reaction. Further, Nanopore DNA sequencing was performed using rapid barcode kit and MinION Mk1B. Results from DNA barcoding of spice samples like Lantana, Four o'clock, Chilli, Ginger, Black pepper, Mustard and Indian medicinal herbs like Ashwagandha, Holy basil will be described. The same methodology can be applied for fungal, animal and insect species identification.
