title: High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping.
abstract: Most mouse genetics laboratories maintain mouse strains that require genotyping in order to identify the genetically modified animals. The plethora of mutagenesis strategies and publicly available mouse alleles means that any one laboratory may maintain alleles with random or targeted insertions of orthologous or unrelated sequences as well as random or targeted deletions and point mutants. Many experiments require that different strains be cross bred conferring the need to genotype progeny at more than one locus. In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products. This requires a large amount of researcher time. Additionally it is susceptible to contamination of future genotyping experiments because it requires that tubes containing PCR products be opened for analysis. Progress has been made with the genotyping of mouse point mutants because a range of new high-throughput techniques have been developed for the detection of Single Nucleotide Polymorphisms. Some of these techniques are suitable for genotyping point mutants but do not detect insertion or deletion alleles. Ideally, mouse genetics laboratories would use a single, high-throughput platform that enables closed-tube analysis to genotype the entire range of possible insertion and deletion alleles and point mutants. Here we show that High Resolution Melt Analysis meets these criteria, it is suitable for closed-tube genotyping of all allele types and current genotyping assays can be converted to this technology with little or no effort.
authors.affiliation: Early Mammalian Development Laboratory, Research School of Biology, The Australian National University, Canberra, Australian Capital Territory, Australia.
authors.forename: Nicole
authors.initials: N
authors.lastname: Thomsen
authors.forename: Radiya G
authors.initials: RG
authors.lastname: Ali
authors.forename: Jehangir N
authors.initials: JN
authors.lastname: Ahmed
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Arkell
mesh_terms: D000483:Alleles; D000818:Animals; D004247:DNA / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D005838:Genotype; D060005:Genotyping Techniques; D057166:High-Throughput Screening Assays; D051379:Mice; D016296:Mutagenesis; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D044366:Transition Temperature
chemical_list: D004247:DNA