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Design and optimization of degenerated universal primers for the cloning of the plant acetolactate synthase conserved domains

Published online by Cambridge University Press:  20 January 2017

Mario Duran Prado ,
Rafael De Prado  and
Antonio R. Franco
Mario Duran Prado
Affiliation:
Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Rabanales, Edificio Severo Ochoa, Córdoba 14071, Spain
Rafael De Prado
Affiliation:
Química Agrícola y Edafología, Universidad de Córdoba, Campus de Rabanales, Edificio Marie Curie, Córdoba 14071, Spain
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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.

Keywords

Cetyltrimethylammonium bromidepigweed quitensis, Amaranthus quitensishairy beggarticks, Bidens pilosacorn poppy, Papaver rhoeascommon waterplantain, Alisma plantago-aquaticarigid ryegrass, Lolium rigidumALS resistanceprimer designPCRherbicide resistanceRAPD
Type
Physiology, Chemistry, and Biochemistry
Information
Weed Science , Volume 52 , Issue 4 , August 2004 , pp. 487 - 491
Copyright
Copyright © Weed Science Society of America 

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