Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays.
We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced-PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced-PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for approximately 1,000 stress-induced and -repressed Arabidopsis genes.
Mahalingam
R
R
Biology Department and Biotechnology Institute, 519 Wartik Laboratory, Pennsylvania State University, University Park, PA 16803, USA.
Fedoroff
N
N
D004251:DNA Transposable Elements; D018744:DNA, Plant / Q000235:genetics; D020224:Expressed Sequence Tags; D015723:Gene Library; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D003313:Zea mays / Q000235:genetics*
D004251:DNA Transposable Elements; D018744:DNA, Plant