Identification of Vernonia zeylanica (Pupula) using DNA barcoding method

Wijayasiriwardena, T.D.C.M.K.¹, De Silva, D.P.C.D.¹, Rathnayaka,R.M.G.S.², Premakumara, G.A.S.¹

1.      Herbal Technology Section, Industrial Technology Institute, 363, Bauddhaloka Mawatha, Colombo 07

2.      Department of Botany, Faculty of Science, University of Kelaniya, Sri lanka.

Objective

To identify Vernonia zeylanica (Pupula)  by using DNA barcoding method.

Abstract

Vernonia zeylanica an endemic medicinal plant which is widely used in Traditional Medicine for fractures and Eczema in Sri Lanka. The plant can be identified by using morphological, microscopical, physico-chemical, phytochemical and DNA barcoding method. DNA barcoding is the latest advanced confirmation test available for the identification of medicinal plants.

In the present study, about 200 mg of two plant samples were taken for genomic DNA extraction .Standard CTAB method with little modifications was used for the extraction and purification. Extracted DNA was amplified using universal primers for matK and rbcL genes in chloroplast genome by PCR (polymerase chain reaction). This works show sequences of these genes are similar within the same species and differ between the different species. Due to the conservation of the sequences within species, matK and rbcL genes together can be used for barcoding and identify the Vernonia zeylanica very accurately. Previous results have been reported the universality of the primers and improved primers can be used for amplification of these genes. Two optimized PCR cycle parameters were used for PCR. Target sequence size for the rbcL primer was about 600 bp and matKA was about 850 bp. Amplified fragments were sequenced and obtained the DNA sequences for the matK and rbcL genes. Sequences were analyzed using Bio edit software package. Sequenced fragments were analyzed and used for DNA barcoding. DNA barcode was submitted to BOLD (Barcoding of Life Database) online database and then uploaded to the `GenBankthrough the BOLD system.

According to the analysis results DNA barcoding method can be considered as the confirmation test for the correct identification of the Vernonia zeylanica

Note: As this is a unpublished paper complete article can't be published at that moment. - Editor-

DNA extraction kits

The DNA Extraction Kit provides a simple, nontoxic method for efficiently isolating high-molecular-weight DNA from tissue, whole blood and cultured cells.

• Simple, non-toxic method for efficiently isolating high-molecular-weight DNA from various sources
• Entire extraction takes only 2 to 3 hours
• No phenol or chloroform required
• DNA isolated is free from contaminants

The extracted and purified DNA is suitable for any molecular biology procedure, including but not limited to PCR (Polymerase Chain Reaction) amplification, restriction digestion, cloning, and sequencing. DNA from various sample types can’t be successfully extracted with same kit. One kit is specific for one kind of samples.

Several kinds of Genomic DNA Purification Kits

Ø  Milk Bacteria DNA Isolation Kit

Ø  Saliva DNA Isolation Kit

Ø  Sputum DNA Isolation Kit

Ø  Fungi/Yeast Genomic DNA Isolation Kit

Ø  Yeast Genomic DNA Isolation Kit

Ø  Bacterial Genomic DNA Isolation Kit

Ø  Stool DNA Isolation Kit

Ø  Phage DNA Isolation Kit

Ø  Urine DNA Isolation Kit

Ø  Dried Blood Spot (DBS) Genomic DNA Isolation Kit

Ø  Blood Genomic DNA Isolation Maxi Kit

Ø  Plant/Fungi DNA Isolation Kit

Kit	protocol
C fast	C Transparent
C High DNA purity	C Modular
C Reliable	C Adaptable
C Reproducible	C Less expensive
D Black box	D Time consuming
D Expensive	D Less reliable
D Low overall yield	D Less reproducible
D Not modular	D No quality guarantee
D Not adaptable	D Need to know how it works

C ^advantages

D ^{disadvantages}