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DNA Fingerprinting and Molecular Marker Development for Baliospermum Montanum (Wïlld.) Muell

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DNA Fingerprinting and Molecular Marker Development for Baliospermum Montanum (Wïlld.) Muell Available online on www.ijppr.com International Journal of Pharmacognosy and Phytochemical Research 2016; 8(8); 1425-1431 ISSN: 0975-4873 Research Article DNA Fingerprinting and Molecular Marker Development for Baliospermum montanum (Wïlld.) Muell. Arg. Lurwan Muazu1, Ramaraj Elangomathavan1, Samiraj Ramesh2* 1Department of Biotechnology, PRIST University, Thanjavur - 614 904, Tamil Nadu, India 2Department of Microbiology, PRIST University, Thanjavur - 614 904, Tamil Nadu, India Available Online: 10th August, 2016 ABSTRACT Baliospermum montanum is one of the medicinal plants, commonly known as ‘Denti’, belongs to the family Euphorbiaceae found in India, Nepal, Burma and Malaya. It has many medicinal values as the pharmaceutical compounds isolated from its secondary metabolites are used for the treatment of various diseases. In the present study, the result shows that the B. montanum genomic DNA extracted, which were randomly amplified using 20 set of primers by arbitrary PCR-based DNA fingerprinting (RAPD) for screening, whereby only 16 primers were able to produce bands at a different score while the remaining three primers remain unamplified. However, out of reproducible primers (MAP- 1-16), three primers (MAP-1, 12 and 14) were found to produce more specific bands, which were selected for specific amplification analysis. Prior to the sequence analysis of specific amplicons (BM MAP-1, BM MAP-13, and BM MAP- 14), the following sequences was retrieved at different size per band, i.e BM MAP-1 (604bp), BM MAP-13 (493bp) and BM MAP-14 (506bp) respectively. However the origin of replication fragments (ORFs) shows BM MAP 01- 604bp has 7 ORFs, BM MAP 13-494bp with 3 ORFs, and BM MAP-506bp has 10 ORFs. In addition, these sequences were submitted to the NCBI website after been analyzed (BLAST) (GenBank accession numbers KX139410.1, KX139411.1 and KX139412.1) that they have no any similarities by the available literature. Thus, could be specifically applicable as a molecular marker of B. montanum DNA fingerprint for identification of medicinal plant adulteration or other molecular analysis. Keywords: Baliospermum montanum; RAPD; DNA sequencing; Molecular marker development INTRODUCTION marker system was absolutely helpful for genetic Baliospermum montanum is belong to the family assessment of plant species, ecological research, for Euphorbiaceae commonly known as ‘Denti’ found in taxonomy and evolutionary. Although some molecular Nepal, Burma, Malaya, and India1, which has so many marker system may principally differ from one another, medicinal value as the pharmaceutical compound isolated but their main function is to express the genome-wide from its secondary metabolite such as codeine, L-dopa, variability4. Therefore molecular markers were used for reserpine, digitalis, and the anticancer drugs vincristine, identification of plant species genetic diversity, as taxol, are used in ovarian and breast cancers treatment2, different types of plant DNA fingerprinting techniques therefore the plant based formulations are utilized by are available5, such as RAPD which is a PCR-based most of the national and international pharmaceutical method of DNA profiling that basically involves the companies for many disorders and disease treatment amplification of DNA sequences using random Primers6. globally2. Their root contained phorbol ester and can also Although the RAPD method uses arbitrary primer be used as an anti-inflammatory while the root past are sequences, many of these primers must be screened in applied to painful swelling and piles. Seeds are used as order to select primers that provide useful amplification powerful hydragogue which promotes watery evacuation products7. of bowels, the seed can also use extensively in rheumatism. However, the seeds are used externally as in MATERIALS AND METHODS snake bite. Leaves used in asthma, abdominal tumor and Plant material bronchitis. The root leaves and the seeds were used The plant was collected from Western Ghats of Tamil Ayurveda in liver disorder, snake bite poisoning and Nadu, in the year June 2013 and preserved in the green abdominal disorder3. Due to the invention of molecular house of parental institution. markers currently, gives a feasible straight evidence in Isolation of genomic DNA relation to genetic divergence as well as the inter- The fresh leaves are collected from green house of grown relationships amongst organisms at their DNA level, B. mantanum, 3g of plant’s leaves were measured and therefore the improvement of a range of molecular cuts into small pieces, then crushed gently using a glass *Author for Correspondence: [email protected] Lurwan et al. / DNA Fingerprinting and… Table 1: DNA finger printing of B. montanum using arbitrary PCR method Primers Primer Sequence No. of No. of Relative Range of Approximate size of (5’- 3’) bases bands Percentage of Amplicons amplicons obtained bands size 380,550,850,950, MAP-1 GTG CAA TGA G 10 07 9.59 380-1800 1100,1300,1800 MAP-2 AGG ATA CGT G 10 02 2.74 400-900 400,900 MAP-3 AAA TCG GAG C 10 04 5.48 375-1200 375,400, 700,1200 MAP-4 AAG ATA GCG G 10 - - - - MAP-5 GGA TCT GAA C 10 04 5.48 700-1700 700,800, 200,1700 MAP-6 TTG TCT CAG G 10 - - - - MAP-7 GTC CTA CTC G 10 01 1.37 750 750 MAP-8 GTC CTT AGC G 10 - - - - 350,700,850,900, MAP-9 TGC GCG ATC G 10 07 9.59 350-1400 1100,1300,1400 MAP-10 AAC GTA CGC G 10 - - - - 500,800,1300,1500,180 MAP-11 GCA CGC CGG A 10 06 8.22 500-3400 0,3400 750,800,1200,1700,220 MAP-12 CAC CCT GCG C 10 05 6.85 750-2200 0 MAP-13 CAT CCC GAA C 10 04 5.48 500-2300 500,900, 1700,2300 MAP-14 GGA CTC CAC G 10 04 5.48 600-1800 600,700, 1200,1800 300,400,600,750, MAP-15 AGC CTG ACG C 10 06 8.22 300-1300 900,1300 MAP-16 CTA TCG CCG C 10 02 2.74 500-750 500,750 350,500,750,1500, MAP-17 CGG GAT CCG C 10 05 6.85 350-1800 1800 300,540,750,800, MAP-18 GCG AAT TCC G 10 06 8.22 300-1500 1400,1500 MAP-19 CCC TGC AGG C 10 04 5.48 400-1600 400,750, 1200,1600 300,400,500,750, MAP-20 CCA AGC TTG C 10 06 8.22 300-1100 900,1100 bead, 3ml of extraction buffer was also added, the entire rpm for 10 min, the upper layer was transferred to another material was transfer into 10 ml polypropylene tube and fresh tube and mixed with a 1/10th volume of 3 M sodium mixed gently. The mixture was incubated for 1 hour at 60 acetate (pH 4.8). 2.5ml of chilled absolute ethanol was 0C in a shaking water bath with intermittent mixing for also added for precipitation. The pellet was dried using each 10 min. A 3ml of chloroform: isoamylalcohol (24:1) air blower then dissolved by addition of 0.5ml of TE was added and mixed gently by inversion for 15 min, buffer. After purification, the DNA isolate was subjected after spins at 8000 rpm for about 10 min at 30 oC, then to electrophoresis for 1 hour at 50V in order to check the upper clear aqueous layer was carefully transferred to their quality, by mixing 2µl of DNA isolate with 2µl of a fresh polypropylene tube. 1.5 ml of 5 M NaCl and an loading dye and loaded onto an agarose gel (0.8% w/v) equal volume of isopropanol (0.6 volume) was added and containing 0.5% of ethidium bromide, and DNA maker mixed gently without any vortex, then was allowed to (1kb DNA ladder) was also loaded to a separate well. stand for 1 hour. The DNA pellet was transferred to 1.5ml Therefore after completing the entire process the gel was microfuge tube, after mixing with isopropanol, the visualized by gel documentation system under UV light, sample was centrifuged at 10,000 rpm for 10 min and which shows a qualitative thick, single band of DNA discarded the supernatant, then the pallet was washed double-stranded with high molecular weight5. with 70% ethanol twice, by adding 0.5ml and centrifuged DNA fingerprint for 5 min at 10,000 rpm, then the supernatant was RAPD Analysis discarded and dried the pallet using an air blower till the RAPD analysis was conducted using 20 random primers entire alcohol smelling was completely disappear. 0.5ml which were arbitrarily selected (MAP-1, MAP- of high salt TE buffer was added to the pallet and mixed 2……MAP-20) (MAP=Medicinal and Aromatic by tapping to dissolve8. plant/primer) for random amplification of DNA DNA purification sequences. The polymerase chain reaction(PCR) were 5µl of DNase was added to the DNA sample and kept in performed based on the following mixture: 8 µl of 2X water bath at 37oC, and allowed to stand for 1 hour of the master mix; primer (map-1 to 20) 1.5 µl; 1µl of DNA incubation period. After incubation, 1ml volume of template and 5.5 µl of double distilled water, therefore chloroform: isoamyl alcohol (24:1) was added and the entire mixture (16 µl) was centrifuged by carefully mixed for 10 min then centrifuged at 10,000 microcentrifuge before the PCR amplification process. IJPPR, Volume 8, Issue 8: August 2016 Page 1426 Lurwan et al. / DNA Fingerprinting and… Table 2: DNA sequences of specific amplified product of modified arbitrary PCR. Sequence Size (bp) Sequence Name 1 GGGTACACCC TCCACTCTCC TCTTCCCCTC CGAATGCCTT CCTGCTAATC CCCTGTGCTC 61 CAATTTCTTT TACTACTGGA ACTTATCCAA CAGAGTTCAC TCAAGGATCG TTCCTAATGG 121 CTCCGGGGCG GGTCAATTCG CCTCCGTCTT CGACGTTCTC CGCTCCTTCC CCTGGCCTTT 181 GCTAGGCCGT GCTGTTCCTC CCCTTCGAGA GCATGTTTGA ATATCTTAAC GTTGTGTTGA 241 CTTATTTGCT GCTTTCGATT AGAGGAAATT TGTGTACTTA TTGTTTGGAT AGCTTTACTT BM MAP 01 604 301 TAGAAAGTAG GGAAAAAACT CAACCGCTAG CAAACAATGG GATTGATTAT ATTACGATCT 361 AATTTAATCC TTTATCGGAC AGGGAAGTTC TCACAATGAA TGTGGAGTTA CCCAGTTATG 421 AGTTACAGTG GGAAAAGAAT ATTAATCTGA ATGCATACTC ATCTCAATCT CTATAAGAGA 481 ATGCTAAACC AATTGTCCTG
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