The novel PCR-based technique of genotyping applied to identification of scrambler mutation in mice.
For a wide range of research purposes it is necessary to perform genotyping, i.e. to test which alleles, each corresponding to a particular locus, are present in the individual genome. Here we suggest a protocol of genotyping for mice with scrambler (scm) mutation. This mutation results in the aberrant splicing of the corresponding mRNA and affects the expression of mdab1 protein. Traditional approaches using genomic Southern hybridization or PCR with specific primers are not suitable for the genotyping of scm because of the lack of comprehensive information on the organization of the gene and on the presence of repetitive sequences in the known region. Here we propose a quick and highly reproducible method for genotyping scm mutant mice. The protocol consists of the following steps: isolation of genomic DNA, digestion with the restriction endonuclease, anchoring of resulting fragments with the adapter, and PCR amplification using adapter-specific primers. The final product of PCR amplification has a characteristic length which is different for the wt (wild type) and scm alleles. Thus, the characteristic pattern of bands obtained for each individual mouse specimen serves as criteria for the presence of wild type and/or scm allele. We believe that this approach could have wider application. The protocol can be easily modified and used as a convenient tool for identification of other genomic defects including those artificially introduced into genome by knockout or gene-trap techniques.
Usman
N
N
Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871 Miklukho-Maklaya 16/10, Moscow, Russia.
Tarabykin
V
V
Gruss
P
P
D000818:Animals; D015139:Blotting, Southern; D002540:Cerebral Cortex / Q000737:chemistry; D017931:DNA Primers; D005838:Genotype; D051379:Mice; D008818:Mice, Neurologic Mutants / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013623:Tail
D017931:DNA Primers; C103773:Dab1 protein, mouse; D009419:Nerve Tissue Proteins