A protocol for high-quality genomic DNA extraction from legumes

Abstract

ABSTRACT. Current DNA extraction protocols, which require liquid nitrogen, lyophilization and considerable infrastructure in terms of instrumentation, often impede the application of biotechnological tools in less researched crops in laboratories in developing countries. We modified and optimized the existing CTAB method for plant genomic DNA extraction by avoiding liquid nitrogen usage and lyophilization. DNA was extracted directly from freshly harvested leaves ground in pre-heated CTAB buffer. Chloroform:isoamyl alcohol (24:1) and RNase treatments followed by single-purification step decontaminated the samples thereby paving way for selective extraction of DNA. High molecular weight DNA yield in the range of 328 to 4776 ng/µL with an average of 1459 ng/µL was obtained from 45 samples of cultivated and wild Cajanus species. With an absorbance ratio at 260 to 280 nm, a range of 1.66 to 2.20, and a mean of 1.85, very low levels of protein and polysaccharide contamination were recorded. Forty samples can be extracted daily at a cost between 1.8 and US$2.0 per plant sample. This modified method is suitable for most plants especially members of the Leguminosae. Apart from Cajanus, it has been extensively applied in DNA extraction from Cicer and Vigna species.

An efficient protocol for total DNA extraction from the members of order Zingiberales- suitable for diverse PCR based downstream applications

“…Using their method, one person is able to process as many as 200 samples in a 5-day working period with a labor cost as low as 100 to US$110 or between 1.8 and US$2 per leaf sample. However in the method reported here, unlike that reported above (Agbagwa et al 2012), no hazardous chemicals such as β-mercaptoethanol, phenol and Rnase were used. In comparison with recently reported method (Agbagwa et al 2012) which took 220 minutes approximately, this method eliminates much of the time consuming steps allowing the whole procedure to be completed within 160 minutes.…”

“…However in the method reported here, unlike that reported above (Agbagwa et al 2012), no hazardous chemicals such as β-mercaptoethanol, phenol and Rnase were used. In comparison with recently reported method (Agbagwa et al 2012) which took 220 minutes approximately, this method eliminates much of the time consuming steps allowing the whole procedure to be completed within 160 minutes. High purity DNA is required for PCR and other PCR-based techniques, such as random amplified polymorphic DNA (RAPD), micro- and macrosatellite analyses, restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) used for genome mapping and DNA fingerprinting (Khanuja et al 1999).…”

“…Previous reports on high-quality plant DNA extraction methods (Aljanabi and Martinez 1997; Zhang and Steward 2000; Karakousis and Langridge 2003; Manen et al 2005; Bokszczanin and Przybyla 2006; Chakraborti et al 2006; Arbi et al 2009; Biswas and Biswas 2011; Japelaghi et al 2011) used liquid nitrogen, lyophilization (freeze-drying), alternating cold (about -80°C), enzymatic digestion for grinding and/or rupturing of the cell and nuclear walls. However, recently, a modified DNA extraction protocol which neither utilizes liquid nitrogen, lyophilization (freeze-drying), alternating cold (about -80°C), nor enzymatic digestion for grinding and/or rupturing of the cell and nuclear walls, has been reported (Agbagwa et al 2012). Using their method, one person is able to process as many as 200 samples in a 5-day working period with a labor cost as low as 100 to US$110 or between 1.8 and US$2 per leaf sample.…”

Biological Role of Trichoderma harzianum-Derived Platelet-Activating Factor Acetylhydrolase (PAF-AH) on Stress Response and Antagonism

“…The genomic DNA of transformants were isolated by a modified CTAB method [22]. In order to identify the PAF-AH deletion transformants: (1) the hyg gene (for hygromycin B resistance) was amplified with primers Hyg-F/Hyg-R; (2) the PAF-AH deletion was verified by attempting to amplify PAF-AH; (3) T-DNA insertion numbers were determined by using primers RB-F/Hyg-R; and (4) primers PAF-AH-UP-F and Hyg-R were used to confirm the hyg cassette position.…”

Characterization of Ty1/copia-like retrotransposon families from pigeonpea genome

“…The seeds of these genotypes were collected from the Indian Institute of Pulses Research, Kanpur, and All Indian Coordinated Research Project on pigeonpea, UAS, Bangalore. Genomic DNA was extracted from the young leaves of 15-day-old seedlings using a modified cetyl trimethyl ammonium bromide method suitable for legumes (Agbagwa et al, 2012 …”