Protocol abstract
Nature Protocols 3, - 1751 - 1765 (2008)
Published online: 23 October 2008 | doi:10.1038/nprot.2008.175
Subject Categories: Genetic analysis | Model organisms | Nucleic acid based molecular biology
Positional cloning by fast-track SNP-mapping in Drosophila melanogaster
Frank Schnorrer1,2,5, Annika Ahlford3,5, Doris Chen1,4,5, Lili Milani3 & Ann-Christine Syvänen3
Abstract
Positional cloning of chemically induced mutations is the rate-limiting step in forward genetic screens in Drosophila. Single-nucleotide polymorphisms (SNPs) are useful markers to locate a mutated region in the genome. Here, we provide a protocol for high-throughput, high-resolution SNP mapping that enables rapid and cost-effective positional cloning in Drosophila. In stage 1 of the protocol, we use highly multiplexed tag-array mini-sequencing assays to map mutations to an interval of 1–2 Mb. In these assays, SNPs are genotyped by primer extension using fluorescently labeled dideoxy-nucleotides. Fluorescent primers are captured and detected on a microarray. In stage 2, we selectively isolate recombinants within the identified 1–2 Mb interval for fine mapping of mutations to about 50 kb. We have previously demonstrated the applicability of this protocol by mapping 14 muscle morphogenesis mutants within 4 months, which represents a significant acceleration compared with other commonly used mapping strategies that may take years.
- Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria.
- Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
- Department of Medical Sciences, Molecular Medicine, Uppsala University, Entrance 70, 3rd floor, Res Department 2, S-75185 Uppsala, Sweden.
- Present address: Department of Biochemistry, Max F. Perutz Laboratories (MFPL), Dr Bohr-Gasse 9/5, A-1030 Vienna, Austria.
- These authors contributed equally to this work.
Correspondence to: Frank Schnorrer1,2,5 e-mail: schnorrer@biochem.mpg.de
Correspondence to: Ann-Christine Syvänen3 e-mail: ann-christine.syvanen@medsci.uu.se
nature-products
A-Z product listing
- 88-mm apple agar plates to collect embryos(Greiner)
- Access to arraying instrument(Cartesian Technologies Inc.)
- Anti-tag oligonucleotides(Integrated DNA Technology)
- Array scanner and software for signal quantification(PerkinElmer Lifesciences)
- Brush to transfer embryos(da Vinci)
- Centrifuge for 96-well microtiter plates(Sorvall)
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