DCC eCatalog

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

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Author(s) :	J. Carroll Bernard W. Graham Michael Findlay Ian Wade Nick Furtado Agnelo R. Searle Iain P. Peace Cameron Waldron Julie,
Publisher :	N/A
Publication Date :	2002
ISSN :	N/A
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 ${\displaystyle 33\; P\; or\; fluorescence\; in\; a\; 30-cycle\; PCR,\; and\; then\; separated\; and\; detected\; on\; large\; polyacrylamide\; sequencing\; gels.\; Highly\; reproducible\; RAF\; markers\; were\; readily\; amplified\; from\; either\; purified\; DNA\; or\; alkali-treated\; intact\; leaf\; tissue.\; RAF\; markers\; typically\; display\; dominant\; inheritance.\; However,\; a\; small\; but\; significant\; portion\; of\; the\; RAF\; markers\; exhibit\; codominant\; inheritance\; and\; represent\; microsatellite\; loci.\; RAF\; compares\; favorably\; with\; AFLP\; for\; efficiency\; and\; reliability\; on\; many\; plant\; genomes,\; including\; the\; very\; large\; and\; complex\; genomes\; of\; sugarcane\; and\; wheat.\; While\; the\; two\; technologies\; detect\; about\; the\; same\; number\; of\; markers\; per\; large\; polyacrylamide\; gel,\; advantages\; of\; RAF\; over\; AFLP\; include:\; (i)\; no\; requirement\; for\; enzymatic\; template\; preparation,\; (ii)\; one\; instead\; of\; two\; PCRs,\; and\; (iii)\; overall\; cost.\; RAF\; and\; AFLP\; were\; shown\; to\; differ\; in\; the\; selective\; basis\; of\; amplification\; of\; markers\; from\; genomes\; and\; could\; therefore\; be\; used\; in\; complementary\; fashion\; for\; some\; genetic\; studies., }$

 

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