title: Detection of low copy number inversions in mouse genomic DNA with unidirectional PCR primers.
abstract: The recessive brachypodism (bp) mutation, located in the growth/differentiation factor 5 (GDF5) gene, causes highly specific skeletal changes in the limbs of brachypod mice. Although Southern blot analysis does not distinguish sequence disruptions in the GDF5 sequence of brachypod mice, sequencing and mapping GDF5 mRNA reveals the bp mechanism to be an inversion preceded by a small deletion. We report here a simple and sensitive method of bp detection from mouse genomic DNA. Previous bp detection used degenerative PCR sequencing. However, without automation, sequencing is a laborious effort for GDF5 inversion detection. The method developed utilizes two unidirectional primers in PCR (UP-PCR), which allow for quick and sensitive analysis of gel electrophoresed PCR products. UP-PCR of the GDF5 gene in wildtype mouse genomic DNA cannot amplify a fragment due to the unidirectional primers. However, UP-PCR of the GDF5 gene in bp mouse genomic DNA does amplify a fragment from the GDF5 gene. Amplification occurs because of the inverted fragment in bp GDF5. This fragment changes the direction of the second forward primer 180 degrees to the position of a reverse primer. UP-PCR detection of the bp inverted fragment is highly sensitive. Amplified fragments were obtained from the bp genomic DNA in the presence of wildtype genomic DNA in ratios up to 1:10(6), respectively. The sensitivity and simplicity of this method allow for quick, inexpensive, and reliable detection of the bp inversion.
authors.affiliation: Department of Biological Sciences, Western Michigan University Kalamazoo 49008, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Lentz
authors.forename: M
authors.initials: M
authors.lastname: Varricchio
authors.forename: S G
authors.initials: SG
authors.lastname: Smith
authors.forename: G
authors.initials: G
authors.lastname: Ficsor
mesh_terms: D000818:Animals; D001483:Base Sequence; D019485:Bone Morphogenetic Proteins; D007446:Chromosome Inversion; D004252:DNA Mutational Analysis / Q000379:methods*; D055428:Growth Differentiation Factor 5; D006133:Growth Substances; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D019485:Bone Morphogenetic Proteins; C525894:Gdf5 protein, mouse; D055428:Growth Differentiation Factor 5; D006133:Growth Substances