The random amplification of polymorphic DNA for fingerprinting plants.

Abstract

School of Biological Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom Molecular markers based upon DNA sequence variation have proved extremely effective tools for distinguishing between closely related genotypes. With the advent of PCR technology, methods have been developed that use amplified DNA sequences as molecular markers. These methods require very little template DNA, which can usually be obtained using a simple minipreparation protocol; the production of marker bands is very fast and far less labor intensive than when using restriction fragment length polymorphism (RFLP) technology. PCR-based techniques have already been used in the analysis of relationships between plants. (1-1~ In this paper, we describe methods for producing plant molecular markers using randomly amplified polymorphic DNA (RAPD) technology. In particular, we describe the effect of varying a range of factors during the procedure and define the protocol that we have found most useful. DNA was extracted from 0.1-0.5 grams of fresh leaf material using a rapid minipreparation method based upon the protocols of Sgai-Maroof et al. (11) and Murray and Thompson.02) Yields of DNA were measured using the Hoechst dye assay method with a TKO 100 Minifluorometer according to the manufacturer's instructions. For PCR, all reaction mixtures had a total volume of 50 ~l. Unless otherwise indicated, the mixture contained 1 unit of Taq polymerase (Cetus), 0.2 nmole (= 4 ~M) of a single 10-mer oligonucleotide, 200 ~M with respect to dATP, dCTP, dTTP, and dGTP, 2.5 mM magnesium chloride, the appropriate dilution of the reaction buffer prepared by the company supplying the polymerase, and not more than 0.1 ~g of plant DNA (see below). All reaction mixtures were overlayed with mineral oil before being placed in a Hybaid Thermal Reactor HBTR1. Unless otherwise indicated, all programs had an initial cycle with 94~ for 7 min, the annealing temperature was 36~ (for 1 rain), and the extension time was 4 min (at 72~ The subsequent 35 cycles were the same except that the 94~ denaturation step was applied for only