Molecular analysis of high-copy insertion sites in maize.
High-copy transposon mutagenesis is an effective tool for creating gene disruptions in maize. In order to molecularly define transposon-induced disruptions on a genome-wide scale, we optimized TAIL-PCR to amplify genomic DNA flanking maize Robertson's Mutator insertions. Sample sequencing from 43 Mutator stocks and the W22 inbred line identified 676 non-redundant insertions, and only a small fraction of the flanking sequences showed significant similarity to maize repetitive sequences. We further designed and tested 79 arbitrary primers to identify 12 primers that amplify all Mutator insertions within a DNA sample at 3.1-fold redundancy. Importantly, the products are of sufficient size to use as substrates or probes for hybridization-based identification of gene disruptions. Our adaptation simplifies previously published TAIL-PCR protocols and should be transferable to other high-copy insertional mutagens.
Settles
A Mark
AM
University of Florida, PO Box 110690, Gainesville, FL 32611-0690, USA. amsettles@mail.ifas.ufl.edu
Latshaw
Susan
S
McCarty
Donald R
DR
D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D018628:Gene Dosage; D017343:Genes, Plant / Q000235:genetics*; D018745:Genome, Plant; D016254:Mutagenesis, Insertional / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D003313:Zea mays / Q000235:genetics*
D017931:DNA Primers; D004251:DNA Transposable Elements