PCR genotyping of oim mutant mice.

A protocol has been developed to distinguish between wild-type, heterozygous and homozygous oim mutant mice. Oim mice (The Jackson Laboratory, Bar Harbor, ME, USA) have a singlebase deletion in their proα2(I) collagen gene (cola-2 gene). This mutation changes the reading frame at the 3′ end of the mRNA causing synthesis of an altered C-propeptide that ultimately inhibits association of these molecules into heterotrimeric type I procollagen bundles in bone matrix (1,2). Bones of homozygous oim mice fracture easily. Thus, they provide a model for moderate to severe osteogenesis imperfecta in humans, also commonly known as brittle bone disease (1). The homozygotes can be identified visually between birth and weaning age by their generally small body stature and obvious swellings and deformities in their limbs and spine caused by bones that break and subsequently heal unset (1). The teeth of homozygotes also break easily. Heterozygous oim mice are visually indistinguishable from wild-type mice. Their body size is normal, and they do not suffer from broken bones when housed under typical research conditions. However, we recently determined that the long bones of oim heterozygotes do not have normal biomechanical characteristics. While not nearly as inferior as homozygous oim bones, they are measurably less strong than those of wild-type mice (Saban et al., unpublished). The protocol described here was developed in our laboratory to distinguish between oim heterozygotes and wildtype mice. It is also used to confirm the identities of homozygous oim animals that receive potentially therapeutic treatments to improve their bone quality. The rationale was to design oligonucleotides that are able to hybridize differentially to the wild-type or mutated allele in the mouse cola-2 gene. Since we must be able to detect a single nucleotide difference between wild-type and mutant alleles, we designed a polymerase chain reaction (PCR) strategy dependent on the differential hybridization of two similar primers and the requirement that the most 3′ nucleotide be base-paired in order for Taq DNA polymerase to extend DNA synthesis from a primer. By running the reactions under very stringent conditions with annealing temperature close to the melting temperature (Tm) of the distinguishing primer, extension from the B21-wt and B21-oim primers only occurs when they are perfectly hybridized. Figure 1 depicts the strategy and placement of PCR primer-cola2 hybrids. Each PCR mixture contains three oligonucleotide primers: F35-wt and B22-wt to generate a 157/158-bp PCR product from either allele (this serves as an internal control) and a B21-wt or B21-oim oligonucleotide to differentiate between the alleles. The distinguishing primers are used in two separate reactions on each genomic DNA sample in order to confirm the genotype of each mouse. The primer sequences are: F35-wt = 5′-GGCTTTCCTAGACCCCGATGCTTAG-3′; B21-oim = 5′-GTCTTGCCCCATTCATTTGTT-3′; B21-wt = 5′-GTCTTGCCCCATTCATTTGTC-3′; B22-wt = 5′-GCAGGCGAGATGGCTTATTTG-3′. Genomic DNA is typically isolated from tail tissue of each mouse. We have also successfully used this method with DNA isolated from blood. Approximately 5–50 ng of genomic DNA is used in amplification reactions in 100 μL volumes containing 10 mM TrisHCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 3 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.27 μM F35-wt primer, 0.35 μM B21oim or B21-wt primer, 0.044 μM B22wt primer and 2.5 U of Taq DNA polymerase. This reaction is overlaid with 50 μL of mineral oil, heated for 5 min at 95°C and subjected to 35 cycles, each having a profile of 60 s at 94°C, 30 s at 64°C and 30 s at 72°C in a Perkin-Elmer DNA Thermal Cycler (Norwalk, CT, USA). Figure 2 shows the DNA banding patterns produced by the three genotypes. F35-wt and B22-wt primers are