After incubation at 95☌ for 3 min the reactions were cycled 30 times: at 94☌ for 30 s, 60☌ for 45 s and 72☌ for 1 min. Ligase mixture consisted of 0.2 µl T4 ligase (New England Biolabs), 0.2 µl 10× ligase buffer, 0.1 µl 100× BSA (New England Biolabs), 0.2 µl 50 mM ATP, 1.2 µl MilliQ (MQ) H 2O and 0.1 µl of enzyme-specific adapter (shown in Table 1) at 50 pmol/µl for MspI and 5 pmol/µl for EcoRI and PstI.Īfter ligation, the mixture was diluted to 500 µl with MQ H 2O and 2 µl used as template in a 50 µl PCR reaction with 2 U of RedTaq polymerase (Sigma) and primers listed in Table 1. After digestion, 2 µl of ligase mixture was added and incubated at 37☌ for 3 h. About 5 ng of DNA from each cultivar was bulked and digested at 37☌ for 1 h with 2 U of restriction enzyme in a volume of 8 µl. Genomic DNA was extracted from young seedlings ( 9). Fragments from representations were cloned and inserts individually amplified from bacterial colonies before arraying on glass slides. Three restriction endonucleases (Table 1) were used to generate representations (below) with complexities reduced by 100–1000-fold compared with total genomic samples. MATERIALS AND METHODS Generating Diversity Panels for riceĭiversity Panels were generated using DNA from nine rice cultivars: Azucena, IR20, IR64, Italica, Bala, Labelle, L203, Millin and Nipponbare. Genetic marker analysis through Diversity Arrays offers a low-cost high-throughput, robust system with minimal DNA sample requirement capable of providing comprehensive genome coverage even in organisms without any DNA sequence information. Here we report the development of a solid-state, open-platform method for DNA polymorphism analysis called Diversity Array Technology, DArT‘. A technology of similar power but with a much greater breadth and much lower cost is needed. SNPs promise to revolutionise biomedicine, but the technology depends on intensive genomic sequencing and a high cost of analysis that cannot be matched in agriculture or basic research. These minor, but abundant, differences in DNA sequence among genotypes are identified through an expensive and laborious DNA sequencing process. For example, DNA chips have been developed to analyse genotypes for single nucleotide polymorphisms (SNPs) ( 8). To overcome many of these restrictions, hybridisation-based methods using nucleic acids immobilised on solid-state surfaces have been developed. Furthermore, all methods based on size separation of multiple DNA fragments suffer from difficulties in precisely correlating bands on gels with allelic variants. Some of these methods (SSR for example) require pre-identification of a polymorphism (or a potential site for it) before analysis of other individuals is possible.

While these genotyping methods have contributed greatly to our current understanding of genome organisation and genetic variation, they are constrained by their dependence on gel electrophoresis, resulting in low throughput. These include restriction fragment length polymorphism (RFLP) ( 1), simple-sequence repeats (SSR) ( 2), random amplified polymorphic DNA (RAPD) ( 3), amplified fragment length polymorphism (AFLP) ( 4), nucleic acid indexing ( 5, 6) and restriction enzyme amplification display system (READS) ( 7). Numerous DNA-based genetic marker analysis methods have been developed over the last two decades. Received NovemRevised and Accepted January 3, 2001. Diversity Arrays enable rapid and economical application of a highly parallel, solid-state genotyping technology to any genome or complex genomic mixtures. The Diversity Panels created using this method allow genetic fingerprinting of any organism or group of organisms belonging to the gene pool from which the panel was developed. Two different approaches are presented: the first involves contrasting two representations on a single array while the second involves contrasting a representation with a reference DNA fragment common to all elements of the array. In the format presented here the technology is assaying for the presence (or amount) of a specific DNA fragment in a representation derived from the total genomic DNA of an organism or a population of organisms. Using the rice genome as a model, we demonstrate the potential of a high-throughput genome analysis method called Diversity Array Technology, DArT‘. Here we present the successful application of the microarray technology platform to the analysis of DNA polymorphisms.

Technology sequence analysis