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Improved PCR-walking for large-scale isolation of plant T-DNA borders.

S. Balzergue,B. Dubreucq,S. Chauvin,I. Le-Clainche,F. Boulaire,R. D. Rose,F. Samson,V. Biaudet,A. Lecharny,C. Cruaud,J. Weissenbach,M. Caboche,L. Lepiniec

Published 2001 in BioTechniques

ABSTRACT

only one sequencing reaction. Instead of using ethanol precipitation after the first PCR, one can use filtration methods in a 96-well format with QIAquick 96 (Qiagen GmbH, Hilden, Germany) or NucleoSpin Multi-96 (Macherey-Nagel GmbH, Düren, Germany). These methods remove the PCR primers more efficiently and resulted in a remarkable reduction of amplification peaks in the intron sequences. However, the quality of exon sequences was the same compared to the purification by ethanol precipitation. The filtration methods are more comfortable in handling but increase the costs per sample. Since both methods revealed good quality of exon sequences, one can choose between the two cleanup methods. Several attempts were made to adapt the EARL strategy to three exons. The reverse primer for exon 9 and the forward primer for exon 15 carried an XbaI restriction site, whereas the reverse primer of exon 15 and the forward primer of exon 16 carried a ClaI site. Multiplex PCR obtained the three amplification products, and ligation was performed after simultaneous XbaI and ClaI restriction. Re-amplification revealed products of expected size in addition to nonspecific smaller fragments, which caused problems in the correct evaluation of the complete sequences of the three exons. Smaller reamplification products presumably result from false ligation. We suggest applying the EARL strategy for only two exons to avoid problems in the evaluation of sequencing data. In summary, by applying the EARL method and a DNA sequencing system for intermediate throughput, we were able to analyze the sequence of four exons on both strands in 96 individuals within one week. This strategy increases the efficiency of the screening of gene polymorphisms and can also be adapted to other genes.

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