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1 July 2004 Design and optimization of degenerated universal primers for the cloning of the plant acetolactate synthase conserved domains
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Abstract

A set of universal and degenerate primers has been designed to clone (by polymerase chain reaction [PCR]) the conserved domains of the acetolactate synthase (ALS) gene where mutations confer resistance to ALS herbicides in plants. These primers were successful in cloning conserved domains of ALS in all monocotyledonous and dicotyledonous plants tested to date, as well as that of bacteria. Total genomic DNA was used as the source of target DNA because no introns were found in the sequences to be amplified. The design of the universal primers was performed after subtle modifications of the consensus degenerate hybrid oligonucleotide primers approach, which implies the synthesis of hybrid oligonucleotide primers containing fixed clamp 5′ and degenerate core 3′ sequences. Optimizations of PCR reactions were done according to a Taguchy approach described for the first time with degenerate oligonucleotides. This method optimizes a PCR reaction using four variables (deoxynucleoside triphosphate, DNA, primers, and Mg2 ) under three different concentrations per variable using just nine reactions. The ALS herbicide-binding domains from many susceptible and resistant plants can be cloned and sequenced in a few hours by using only 100 mg of starting plant material, like one leaf or several small seedlings or seeds.

Nomenclature: Cetyltrimethylammonium bromide; pigweed quitensis, Amaranthus quitensis; hairy beggarticks, Bidens pilosa; corn poppy, Papaver rhoeas; common waterplantain, Alisma plantago-aquatica; rigid ryegrass, Lolium rigidum.

Mario Duran Prado, Rafael De Prado, and Antonio R. Franco "Design and optimization of degenerated universal primers for the cloning of the plant acetolactate synthase conserved domains," Weed Science 52(4), 487-491, (1 July 2004). https://doi.org/10.1614/WS-03-098R
Received: 25 June 2003; Accepted: 1 October 2003; Published: 1 July 2004
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