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.
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Supplementary Data | An Excel file with example SNPmapper input files. Explanatory comments of the format are provided. (XLS 58 kb)
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Schnorrer, F., Ahlford, A., Chen, D. et al. Positional cloning by fast-track SNP-mapping in Drosophila melanogaster. Nat Protoc 3, 1751–1765 (2008). https://doi.org/10.1038/nprot.2008.175
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DOI: https://doi.org/10.1038/nprot.2008.175
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