Journal: PLoS Genetics
Article Title: MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice
Figure Lengend Snippet: Representative CAG repeat distributions, and Msh3 variations in B6 and CBy mice. A) The autoradiographs show representative SP-PCR analyses of DNA, extracted from heart, liver, striatum and tail. At weaning the B6.Cg-R6/1 (B6) and CBy.Cg-R6/1 (CBy) congenic mice contained in tail DNA (CAG)98 and (CAG)94, respectively. For comparison the profiles of the Msh2 −/− mouse is shown. About 5–10 DNA amplifiable molecules were amplified in each reaction with primers MS-1F and MS-1R. Animals were 20-weeks old. B) Congenic CBy.Cg-R6/1 mice were crossed to B6 and the resulting F1 progeny were crossed to produce F2 mice with all possible genotypes at the Msh3 locus. Repeat instability was assayed by amplifying 10 ng genomic DNA using fluorescently labelled primers and resolving the fragments by capillary gel electrophoresis ( Figure 1B ). Using this high-resolution approach repeat length distributions present with the typical ‘hedgehog’ pattern ( e.g.  ,  ,  ,  . This pattern reflects both somatic mosaicism within the sample and PCR artefacts generated by Taq polymerase slippage  ,  . The PCR artefacts are predominantly repeat contractions, hence these are not considered here. The pattern of CAG repeat instability depended on genotype at the MSH3 locus. B6 homozygosity resulted in the greatest instability, CBy homozygosity resulted in lack of expansion, while heterozygosity resulted in an intermediate instability, indicative of a gene dosage effect of the Msh3 locus. Numbers indicate the CAG repeat size corresponding to major peaks. In addition, on the B6 tracing, a second number indicates the highest CAG repeat number detected. C) Msh3 polymorphisms in Msh3 gene from C57BL/6 (B6) and BALB/cBy (CBy) mice. Promoters were identical. SNPs were identified or confirmed to those in dbSNP by sequencing the Msh3 gene.
Article Snippet: Qiagen Taq polymerase (cat #201225) was used as recommended by the manufacturer.
Techniques: Mouse Assay, Polymerase Chain Reaction, Amplification, Mass Spectrometry, Nucleic Acid Electrophoresis, Generated, Sequencing