RAPD analysis of mtDNA from tomato flowers free of nuclear DNA artifacts.

BENCHMARKS Random amplified polymorphic DNA (RAPD) is widely used with whole cell DNA to determine genetic diversity among plants. The method is also useful when applied specifically to mitochondrial DNA (mtDNA) for plant genotyping and for characterization of organelle-associated changes such as cytoplasmic male sterility (CMS; References 1–3). Some have found that mitochondrial-specific RAPD is more sensitive than mitochondrial restriction fragment length polymorphism (RFLP) (1). However, its general use is limited because of the difficulty in obtaining mtDNA that is free of contaminating nuclear DNA without using laborious gradient ultracentrifugation procedures or large amounts of the DNase I (4). Here we demonstrate a simple and inexpensive mtDNA isolation procedure from flower tissue that results in highly reproducible mitochondrial-specific RAPD profiles without nuclear DNA artifacts. The method combines a differential centrifugation protocol (5) with a DNase I treatment that is optimized by assessing the amount of nuclear DNA contamination by PCR. For each isolation, we homogenized 5 g of tomato flower tissue, collected without the sepals and stored at-80°C, in high ionic-strength buffer [50 mM Tris-HCl, pH 8.0, 1.3 M NaCl, 25 mM EDTA, 0.2% bovine serum albumin (BSA), and 0.05% cysteine and 56 mM β-mercaptoethanol added before use; 5 mL/g tissue] and centrifuged it three times as previously described (2600× g for 15 min; 2600× g for 10 min; and finally , 14,500× g for 15 min to pellet the mitochondria; Reference 5). To eliminate nuclear DNA, the mitochondrial pellet was resuspended in 980 μL of cold DNase I buffer (50 mM Tris-HCl, pH 7.5, and 10 mM MgCl 2) and transferred to a 1.5-mL microcentrifuge tube (Fisherbrand 1 mg/mL DNase I in a buffer containing 50% glycerol, 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl 2 , and 1 mM 1,4 dithiothreitol were added, and the tube was incubated at 37°C for 1 h. The reaction was stopped with the addition of 50 μL of 0.5 M EDTA, 5.25 mg/mL sodium dodecyl sulfate (SDS), and 0.1 volume of 1 M Tris-HCl, pH 8.0. The mtDNA was then extracted with phenol/chloroform/iso-amyl alcohol (25:24:1), pH 6.7 (Fisher Chemicals , Fairlawn, NJ, USA), and water-saturated chloroform. The supernatant was precipitated at-20°C with 0.3 M NH 4 acetate and 2 volumes of cold 100% ethanol overnight. The DNA was pelleted by centrifuga-tion at 16,000× g for 30 min and resuspended in 50 μL of TE (Tris-EDTA) buffer. RNA was removed …