Economical and rapid method for extracting cotton genomic DNA.

Abstract

A fast, simple, and reliable mini-prep method for the extraction of DNA from Gossypium species and cultivars has been developed. This small-scale method is cetyltrimethylammonium bromide (cTAB)-based, and it extracts DNA from one to three folded or nearly unfolded young leaves processed in a 1.5 mL tube with 0.5 mL of extraction buffer and homogenized by an electric drill. Compared with the macro-prep cTAB method, the improved mini-prep method is highly efficient and much cheaper in terms of time, chemical use, and labor input. Easily 200 samples per day can be processed by a single person. The DNA yield is greater (60 )g per 50-100 mg of fresh leaf tissue) than that obtained from the macroprep method (50 )g from 5 g of fresh leaf tissue) and it provides DNA for 3000 to 6000 polymerase chain reactions (PCRs). The DNA quality is sufficient for PCR-based and endonuclease restriction marker analysis. With the development of polymerase chain reaction (PCR) technology, molecular markers J. Zhang and J.McD. Stewart, 115 Plant Sci. Bldg., Dep. of Crop, Soil, and Environ. Sci., Univ. of Arkansas, Fayetteville, AR 72701. Received 10 Mar. 2000. *Corresponding author ([e-mail]). Abbreviations: PCR, polymerase chain reaction; RAPD, random amplified polymorphic DNA; SSR, simple sequence repeat; AFLP, amplified fragment length polymorphism; RFLP, restriction fragment length polymorphism; cTAB, cetyltrimethylammonium bromide. 194 ZHANG & STEWART: ECONOMICAL, RAPID GENOMIC DNA EXTRACTION based on PCR soon found vast application in plant genetics and breeding (Lee, 1995). To accommodate the need for PCR-based markers, a rapid, simple, and reliable DNA preparation method is required to provide high quality and quantity DNA for the analyses. Although numerous DNA extraction methods for plants have been reported in the literature, the cTAB extraction method is used most often. The traditional macro-preparation of DNA usually requires from 0.5 to several grams of plant tissue, making it impractical to analyze individual plants during early seedling stage. Also, the methods are time consuming and laborious due to their multistep procedures. Furthermore, large amounts of hazardous chemical solvents are required. Modifications have been made for plant species such as cotton that are high in polysaccharides and polyphenols. The compounds form a sticky, brown gelatinous matrix during DNA preparation that interferes with DNA digestion and PCRs. These modified methods usually employ high salt concentrations to remove polysaccharides, and polyvinylpyrrolidone to bind polyphenols (Lodhi et al., 1994; Porebski et al., 1997). Ascorbic acid, emercaptoethanol, and activated charcoal were found to improve extracted DNA quality (Paterson et al., 1993; Bi et al., 1996). For PCR-based DNA markers used in markerassisted selection, a fast DNA extraction method is needed. A reliable method should meet the following criteria: (i) require only a small amount of tissue; (ii) involve simple procedures; (iii) use minimal number and amount of chemicals; (iv) yield high-quality DNA; and (v) yield large quantities of DNA. Many mini-prep methods for obtaining DNA have been developed that have included such modifications as no grinding, no centrifugation, and/or no liquid transfer.