2017 Vol.23 No.1 PP 25-36

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

N.NANDHA KUMAR¹, ^*K. SOURIANATHA SUNDARAM¹, D. SUDHAKAR² AND K.K. KUMAR³

¹Department of Fruit Crops,
²Department of Plant Biotechnology,
Horticultural College and Research Institute,
Tamil Nadu Agricultural University,
COIMBATORE-641 003 (T.N.) INDIA
³Plant Breeding and Genetics,
Agricultural College & Research Institute,
Killikulam, TUTICORIN -628 252 (T.N.) INDIA
*Corresponding Author E-mail: sooria@tnau.ac.in Department of Biotechnology,
Dr. M.P.S. Group of Institutions,
Dr. B.R. Ambedkar University,
AGRA (U.P.) INDIA *Corresponding Author

ABSTRACT

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

Key words : CTAB, DNA extraction, Musa spp, Octanol, Proteinase K, PVP.