Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.
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 x 10(10) 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.
Liu
Y G
YG
Mitsui Plant Biotechnology Research Institute, Tsukuba Laboratory, Japan.
Mitsukawa
N
N
Oosumi
T
T
Whittier
R F
RF
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D013816:Thermodynamics
D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C036483:T-DNA