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  • HortFlora Research Spectrum
  • HortFlora Research Spectrum
  • HortFlora Research Spectrum
  • HortFlora Research Spectrum
  • HortFlora Research Spectrum
Journal : HortFlora Research Spectrum
Volume : Volume 6, Issue 3: September 2017
Page (s) : 159-162
Date of Publication
Print :
25-Sep-2017
Article Type : Full Length Original Article
Online : 25-Sep-2017
Title:
Standardization of Phenol Free Genomic DNA Extraction of Pomegranate Genotypes for Diversity Analysis
Author(s):
Dimpy Raina* and A. S. Sundouri
*Corresponding Author's E-mail :
dimpy.raina@gmail.com

Abstract

The isolation of intact high-molecular-mass genomic and good quality deoxyribonucleic acid (DNA) is the pre-requisite for many molecular biology applications including long polymerase chain reaction (PCR), endonuclease restriction digestion, southern blot analysis, and genomic library construction. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in pomegranate leaves poses problem in getting good quality DNA. The study aimed to determine a reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from pomegranate leaves. Easy purification method was added to modify CTAB method using Tris-saturated phenol: chloroform (1:1) and 3M sodium acetate. Polyvinylpyrrolidone (PVP) and -mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment. Efficient yields of high-quality amplifiable DNA (200-1200 ng) was produced rapidly with modified CTAB method. Quantity of obtained DNA from this extraction method was controlled in terms of absorbance at wavelength of 260, 230 and 280 nm. The absorbance ratio of A260/A280 indicates presence of dense protein. Spectrophotometric analysis at A260/A280 revealed ratio range of 1.77–1.94. The purified DNA which has excellent spectral quality was efficiently amplified by 48 SSR primers and was suitable for long-fragment PCR amplification.
 

Cite this article as:
Raina D. and Sundouri A. S. (2017). Standardization of phenol free genomic DNA extraction of pomegranate genotypes for diversity analysis. HortFlora Res. Spectrum, 6(3) : 159-162.
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