Detection of low copy number inversions in mouse genomic DNA with unidirectional PCR primers

✍ Scribed by S. J. Lentz; M. Varricchio; S. G. Smith; G. Ficsor

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 75 KB
• Volume: 30
• Category: Article
• ISSN: 0893-6692
• DOI: 10.1002/(sici)1098-2280(1997)30:3<260::aid-em3>3.0.co;2-p

✦ Synopsis

The recessive brachypodism (bp) mutation, located UP-PCR of the GDF5 gene in wildtype mouse genoin the growth/differentiation factor 5 (GDF5) gene, mic DNA cannot amplify a fragment due to the unicauses highly specific skeletal changes in the limbs directional primers. However, UP-PCR of the GDF5 of brachypod mice. Although Southern blot analysis gene in bp mouse genomic DNA does amplify a does not distinguish sequence disruptions in the fragment from the GDF5 gene. Amplification occurs GDF5 sequence of brachypod mice, sequencing because of the inverted fragment in bp GDF5. This and mapping GDF5 mRNA reveals the bp mecha-fragment changes the direction of the second fornism to be an inversion preceded by a small dele-ward primer 180Њ to the position of a reverse tion. We report here a simple and sensitive method primer. UP-PCR detection of the bp inverted fragof bp detection from mouse genomic DNA. Previous ment is highly sensitive. Amplified fragments were bp detection used degenerative PCR sequencing. obtained from the bp genomic DNA in the presence However, without automation, sequencing is a labo-of wildtype genomic DNA in ratios up to 1:10 6 , rious effort for GDF5 inversion detection. The respectively. The sensitivity and simplicity of this method developed utilizes two unidirectional prim-method allow for quick, inexpensive, and reliable ers in PCR (UP-PCR), which allow for quick and sen-detection of the bp inversion. Environ. Mol. Mutasitive analysis of gel electrophoresed PCR products. gen.