Positional cloning by fast-track SNP-mapping in Drosophila melanogaster

Nat Protoc. 2008;3(11):1751-65. doi: 10.1038/nprot.2008.175.

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.

MeSH terms

  • Animals
  • Chromosome Mapping / methods*
  • Cloning, Molecular / methods*
  • Drosophila melanogaster / embryology
  • Drosophila melanogaster / genetics*
  • Embryo, Nonmammalian
  • Genetic Markers
  • Genotype
  • Nucleic Acid Hybridization
  • Oligonucleotide Array Sequence Analysis
  • Phenotype
  • Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide*
  • Recombination, Genetic
  • Software

Substances

  • Genetic Markers