Journal: The Journal of Molecular Diagnostics : JMD
Article Title: A Pseudo-Full Mutation Identified in Fragile X Assay Reveals a Novel Base Change Abolishing an EcoRI Restriction Site
Figure Lengend Snippet: Determination of the origin of the 10.9-kb fragment from the index patient. A: Southern blot using PstI digestion only showing bands of 0.99, 1.03, and 1.08 kb (the largest band approximately 1.1 kb in size). An abnormal control (AC, lane 6 ) shows a full mutation (Coriell GM07537A), with an additional 2.1-kb band (336 CGG repeats). No CGG expansion was noted in the family study ( lanes 2 to 4 ) or in a normal control (NC, lane 5 ). MW, the Lambda/HindIII MW marker (Chemicon International). B: Southern analysis of the family digested with EcoRI, with (+) and without (−) NruI. MW, molecular marker as in A ; NFC, normal female control; M, mother; IP, index patient; F, father; NMC, normal male control; AC, abnormal control as in A . Note that father and the index patient both showed the 10.9-kb fragment ( lanes 6 , 7 , and 9 ). C: PCR products with (+) and without (−) EcoRI digestion. MR, 100-bp Molecular Ruler (Bio-Rad); NC, normal control; M, mother; IP, index patient; F, father.
Article Snippet: For the standard Southern analysis, 6 μg of genomic DNA was double-digested with NruI (a methylation-sensitive enzyme, cutting only unmethylated, active X chromosome) and EcoRI (100 U of each enzyme at 37°C for 6 hours), electrophoresed on a 0.7% agarose gel, transferred to a nylon membrane, and hybridized with digoxiginin-labeled probe pFXa1NHE (Chemicon International, Temecula, CA).
Techniques: Southern Blot, Mutagenesis, Marker, Polymerase Chain Reaction