Species specific DNA sequences and their utilization in identification of Viola species and authentication of "banafsha" by polymerase chain reaction
AssigneeCouncil of Scientific and Industrial Research
InventorsMahipal Singh, Chandan Sharma, Brij Lal
Life of this patent
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Abstract
This invention relates to Viola specific DNA sequences and methods for identification of Viola species using the said sequences. The invention also relates to a method for detection of adulteration in ‘banafsha’ by polymerase chain reaction method. Particularly this invention relates to a very sensitive, accurate and efficient method for identification of Viola species from even the powdered form of the plant or its admixtures with other plant species. More particularly, the method is designed to test the genuineness of ‘banafsha’.
Claims14
DNA sequences specific to Viola species selected from the group comprising Viola odorata, V. canescens, V. pilosa, V. betonicifolia, and V. tricolor obtained from their 5S rRNA gene spacer region comprising:
- 5 S. Primer Total N. code Sequence bases 1. BT61.F 5′---GGTGAGAACTCTCGAGGGTCGGG 24 A---3′ 2. BT61.R 5′---GCCCCGATCCGACACCCGAGC---3′ 21 3. BT101.F 5′---CCCTCACTCCTCGAGAATATG---3′ 21 4. BT91.F 5′---CTATTTACTTCTCTCACCGCG---3′ 21 5. BT811.F 5′---TTTGTAAACACGGAGGGGGC-3′ 20 6. BT811.R 5′---ACAAACCCACGATTGGATTG---3′ 20 7. BT71.F 5′---TTGTAAACACAGAGGAGGG---3′ 19 8. BT71.R 5′---CACGATTGGATTACACGC---3′ 18
A method for the detection and identification of Viola species specific sequence from a sample, said method comprising the steps of:
- a) amplifying 5S rRNA gene spacer region from Viola species with consensus forward (M27) and reverse (M28) primers by polymerase chain reaction,
- b) analysing the PCR products on agarose gel and identifying the lowest densely visible band in each species under UV light,
- c) cloning the above identified band in an appropriate vector and transforming in E-coli MoS blue cells,
- d) analysing the clones for the right insert by isolating the plasmid DNA from transformed cells,
- e) sequencing the selected pDNA clones of all 5 species of Viola in both directions using T7 promoter primer and M13 forward primer, and
- f) analysing the sequenced data and designing species specific primer to Viola species.
Dependencies are inferred from the claim text.