PCR template-DNA isolated quickly from monocot and dicot leaves without tissue homogenization.

Abstract

Nucleic Acids Research, 1994, Vol. 22, No. 10 PCR template-DNA isolated quickly from monocot and dicot leaves without tissue homogenization Christie E.Williams* and Pamela C.Ronald Department of Plant Pathology, University of California, Davis, CA 95616, USA Received April 5, 1994; Accepted April 15, 1994 * To whom correspondence should be addressed for 5 min. Hot samples were transferred directly to a speed-vac and vacuum infiltrated for 2 min (rice, bean, corn and barley) or 6 min (oat, triticale, wheat and durum). After treatment, the leaf tissue was somewhat translucent and the buffer noticeably a. kb b. kb C. kb m 1 2 3 4 5 6 Figure 1. Separation of PCR products on gels containing 1 % Synergel (Diversified Biotech) and 0.6% agarose (BRL) in 0.5x TBE buffer. A) Lane 1 and 2 contain DNA amplified from large-scale isolations of parental plant DNA. Subsequent lanes contain DNA amplified from PEX isolations of DNA from F 2 plants. Amplification with 24-base STS primers B7 and B8 (7) resulted in single PCR products from parental classes of homozygotes (lanes 1, 2 and 6) and the two parental plus one heteroduplex product from heterozygotes (lanes 3, 4 and 5). The heteroduplex is a PCR artifact that does not migrate in accordance with its actual molecular weight (12). B) Lanes are in the same order as those in A. Amplification with 10-base RAPD primer A02 (Operon, Inc.) produced no polymorphisms among the samples. C) RAPD primer AB09 generated discrete amplification products from bean (lane 1), barley (lane 2), oat (lane 3), com (lane 4), rice (lane 5) and triticale Oane 6). Lane m contains 1 kb ladder. Downloaded from nar.oxfordjournals.org at University of California, Davis on October 26, 2010 A protocol is presented that utilizes potassium ethyl xanthogenate to liberate DNA from leaves without the need for tissue homogenization. This quick, single-tube method requires only 1/3 cm 2 of tissue and yields enough DNA to serve as template for 20 polymerase chain reactions. The resulting amplification patterns are indistinguishable from those generated from rigorous large-scale DNA extractions. Because 100—200 samples can be processed per day by a single person, this method allows for the rapid screening of large mapping populations. This protocol was developed for use with rice, but also works well with other species including barley, oat, corn and bean. Although many quick protocols have been published for small- scale isolation of DNA, certain characteristics of rice and other monocots result in inconsistent yields and poor amplification with the polymerase chain reaction (PCR). Because rice leaves are very fibrous, protocols specifying homogenization of leaf tissue (1) are laborious and undependable when scaled down. Quick protocols that amplify DNA directly from leaf tissue (2, 3, 4) have failed to rupture the tough cell wall of rice leaves sufficiently to liberate template DNA. Fast methods for the extraction of DNA from single seeds of cotton (5) rye and barley (6) and rice (7) have been published. In our hands, these procedures did yield sufficient quantities of rice DNA for PCR (about 100 ng per half- seed). However, PCR amplification was inconsistent, presumably due to the presence of inhibitors like starch. As a result, we designed a quick, small-scale prep that utilizes potassium ethyl xanthogenate (PEX; 8) to dissolve cell walls, degrade proteins and inhibit DNase activity (8). No tissue homogenization is required in order to yield enough DNA for 20 PCR amplifications from a small amount of leaf tissue. We tested our protocol on young leaves from bean (Phaseolus vulgaris), barley (Hordeum vulgare), corn (Zea mays), oat (Avena sativa), rice (Oryza sativa), triticale (a wheat/rye hybrid), wheat (Jriticum aestivum) and durum (T.durum). Upon emergence from the soil, the first true leaf was collected on ice in the greenhouse and subsequently stored at -80°C. Older rice tissue, up to 8 weeks after emergence, also was collected. Frozen segments of leaf totalling 1/3 cm 2 were selected and placed in the bottom of a small microcentrifuge tube containing 100 /tl of extraction buffer (8). This buffer consisted of 6.25 mM PEX (potassium ethyl xanthogenate or carbonodithioic acid, o-ethyl, potassium salt from Fluka), 100 mM Tris-HCl (pH 7.5), 700 mM NaCl, 10 mM EDTA (pH 8). Dipping the hydrophobic leaf tissue in 70% ethanol prior to placing it in buffer helped to keep it fully submerged. The samples were incubated in a water bath at 65 C C