Table of Contents Author Guidelines Submit a Manuscript
Journal of Biomedicine and Biotechnology
Volume 2 (2002), Issue 3, Pages 141-150
http://dx.doi.org/10.1155/S1110724302206026
Research article

Randomly Amplified DNA Fingerprinting: A Culmination of DNA Marker Technologies Based on Arbitrarily-Primed PCR Amplification

1Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane, Queensland 4072, Australia
2School of Land and Food Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia
3CSIRO Division of Plant Industry, 306 Carmody Road, St Lucia, Queensland 4067, Australia
4Bureau of Sugar Experiment Stations, Indooroopilly, Queensland 4068, Australia
5Queensland Agricultural Biotechnology Centre, Gehrmann Labs, St Lucia, Queensland 4072, Australia
6Australian Genome Research Facility, Gehrmann Labs, St Lucia, Queensland 4072, Australia

Received 25 June 2002; Accepted 30 June 2002

Copyright © 2002 Hindawi Publishing Corporation. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Arbitrarily-primed DNA markers can be very useful for genetic fingerprinting and for facilitating positional cloning of genes. This class of technologies is particularly important for less studied species, for which genome sequence information is generally not known. The technologies include Randomly Amplified Polymorphic DNA (RAPD), DNA Amplification Fingerprinting (DAF), and Amplified Fragment Length Polymorphism (AFLP). We have modified the DAF protocol to produce a robust PCR-based DNA marker technology called Randomly Amplified DNA Fingerprinting (RAF). While the protocol most closely resembles DAF, it is much more robust and sensitive because amplicons are labelled with either radioactive 33P or fluorescence in a 30-cycle PCR, and then separated and detected on large polyacrylamide sequencing gels. Highly reproducible RAF markers were readily amplified from either purified DNA or alkali-treated intact leaf tissue. RAF markers typically display dominant inheritance. However, a small but significant portion of the RAF markers exhibit codominant inheritance and represent microsatellite loci. RAF compares favorably with AFLP for efficiency and reliability on many plant genomes, including the very large and complex genomes of sugarcane and wheat. While the two technologies detect about the same number of markers per large polyacrylamide gel, advantages of RAF over AFLP include: (i) no requirement for enzymatic template preparation, (ii) one instead of two PCRs, and (iii) overall cost. RAF and AFLP were shown to differ in the selective basis of amplification of markers from genomes and could therefore be used in complementary fashion for some genetic studies.