Development of a High Quality, Rapid, Efficient and Economical DNA Extraction Protocol from Climate Resilient Pearl Millet Crop Without Liquid Nitrogen

Abstract

Extraction of good quality genomic deoxyribonucleic acid (DNA) from plants is a major prerequisite for molecular biology experiments. An efficient genomic DNA protocol must be simple, fast and cost effective with high yield and purity. Presence of polyphenols, polysaccharides and secondary metabolites in some plants hamper with DNA extraction making it a very laborious, difficult and time consuming procedure. Here, we portrayed a modified protocol based on the cetyl trimethyl ammonium bromide (CTAB) method to isolate DNA from climate resilient pearl millet leaf tissues having higher amount of polysaccharides. It also excludes the use of expensive chemicals and equipments like proteinase K, liquid nitrogen and tissue lyser. This method includes extraction of DNA using a buffer (pH 8.0) containing 200 mM Tris-HCl, 20 mM ethylenediamine tetracetic acid (EDTA),1.4 M NaCl, 2% CTAB, 2% sodium dodecyl sulphate (SDS) and 1.0 % β-mercaptoethanol followed by purification of DNA with phenol, chloroform, isoamyl alcohol and finally precipitation of DNA by sodium acetate and isopropanol. Good quality genomic DNA with sharp and clear bands was obtained from 48 pearl millet genotypes using this protocol. The yield of DNA varied from 105.2 to 328.3 ng/μl. The purity of DNA sample ranged from 1.74 to 1.95 based on the absorbance at A260/A280 ratio indicating that it’s free from ribonucleic acid (RNA) and protein contamination. PCR analysis using simple sequence repeat (SSR) primers exhibited consistent and reliable amplification products ranging from 150 to 650 bp.This study reveals a fast, simple, efficient, specific, reproducible, reliable and cost effective method for extraction of DNA from small to large number of plant samples amenable to PCR amplification and could be stored for longer duration.

Detection of True Hybrids in Pearl Millet Cross Combinations by Employing SSR Molecular Markers

“…The amplification process began with an initial denaturation phase at 94°C for 5 minutes, succeeded by 35 cycles. Each cycle consisted of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and primer extension at 72°C for 1 minute, as previously outlined by Ambawat et al [47]. A final extension step was conducted for 10 minutes at 72°C, succeeded by a holding period at 4°C.…”

Molecular Characterization of Drought Tolerant Genotypes of Pearl Millet [Pennisetum glaucum (L.) R. Br.] for A1 Zone

“…Genomic DNA was extracted from young and fresh leaves of 12 days old plantlets of 24 genotypes using cetyl trimethyl ammonium bromide (CTAB) method along with some modifications as reported in our other study [11]. 2-3 leaves from each genotype were ground in a pestle and mortar without liquid nitrogen and the mixture was transferred into 2 ml eppendorf tubes and 1200 μl of CTAB extraction buffer was added to them.…”