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Efficient isolation and mapping of Arabidopsis thaliana T‐DNA insert junctions by thermal asymmetric interlaced PCR

Efficient isolation and mapping of Arabidopsis thaliana T‐DNA insert junctions by thermal... Thermal asymmetric interlaced (TAIL‐) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones. Highly specific amplification is achieved without resort to complex manipulations before or after PCR. The adaptation of this method for recovery and mapping of genomic sequences flanking T‐DNA insertions in Arabidopsis thaliana is described. Insertion‐specific products were amplified from 183 of 190 tested T‐DNA insertion lines. Reconstruction experiments indicate that the technique can recover single‐copy sequences from genomes as complex as common wheat (1.5 × 1010 bp). RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T‐DNA transgenic lines were determined on an RFLP map. These lines, whose mapped T‐DNA insertions confer hygromycin resistance, can be used for fine‐scale mapping of linked phenotypic loci. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Plant Journal Wiley

Efficient isolation and mapping of Arabidopsis thaliana T‐DNA insert junctions by thermal asymmetric interlaced PCR

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Publisher
Wiley
Copyright
Copyright © 1995 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0960-7412
eISSN
1365-313X
DOI
10.1046/j.1365-313X.1995.08030457.x
Publisher site
See Article on Publisher Site

Abstract

Thermal asymmetric interlaced (TAIL‐) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones. Highly specific amplification is achieved without resort to complex manipulations before or after PCR. The adaptation of this method for recovery and mapping of genomic sequences flanking T‐DNA insertions in Arabidopsis thaliana is described. Insertion‐specific products were amplified from 183 of 190 tested T‐DNA insertion lines. Reconstruction experiments indicate that the technique can recover single‐copy sequences from genomes as complex as common wheat (1.5 × 1010 bp). RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T‐DNA transgenic lines were determined on an RFLP map. These lines, whose mapped T‐DNA insertions confer hygromycin resistance, can be used for fine‐scale mapping of linked phenotypic loci.

Journal

The Plant JournalWiley

Published: Sep 1, 1995

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