Positional cloning by fast-track SNP-mapping in Drosophila melanogaster
2008 (English)In: Nature Protocols, ISSN 1754-2189, Vol. 3, no 11, 1751-1765 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2008. Vol. 3, no 11, 1751-1765 p.
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-98390DOI: 10.1038/nprot.2008.175ISI: 000265781600008PubMedID: 18948975OAI: oai:DiVA.org:uu-98390DiVA: diva2:174348