From sequencing is not possible in differential
From the literature review conducted, many problems were identified with DNA barcode for species specific unique identification as it had several limitations in mixed, highly processed powdered or liquid samples. When the plant materials are mixed as powdered or in liquefied form applications of DNA barcodes based on DNA sequencing is not possible in differential detection of plant species. The requirement of a possible method which directly identifies the uniqueness of a species has occurred. Therefore species specific PCR based method could be used to overcome this problem. In this chapter an accurate technique, insilico species specific primer designing by using DNA barcode regions has been discussed and for the efficiency of this process, automated application to generate species specific unique primers from the DNA barcode regions were designed.
1.1. Species specific primer designing using DNA barcode regions
During this study, I have selected medicinal plants and its possible adulterant/substituent species as the sample sequences to develop species specific primers.
1.1.1. Retrieve DNA barcode sequences
22.214.171.124. Identification of DNA barcode regions of plants
According to the previous studies, many DNA barcode regions were identified (Table 2). ITS1/ ITS2 were used as nuclear ribosomal DNA barcode regions and matK, rbcL as plastid DNA barcode regions and trnH-psbA as non-coding intergenic spacer region. These regions were used as sequences for specific primers design to determine internal variations.
126.96.36.199. Selection of DNA barcode primers
A collection of universal pant barcode primers from several types of researches was used to select the appropriate DNA barcode regions to find internal sequence variations for species specific primer design.