Neurogenetics : methods and protocols by N. Potter

By N. Potter

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By N. Potter

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Heerema, N. , Dlouhy, S. , Vance, G. , and Vogt, P. H. (1997) Multiplex PCR: Critical Parameters and Step-by-Step Protocol. BioTechniques 23, 504 – 511. FMR1 Trinucleotide Repeat Expansion Mutations II TRINUCLEOTIDE REPEAT DETECTION 27 FMR1 Trinucleotide Repeat Expansion Mutations 29 3 Detection of FMR1 Trinucleotide Repeat Expansion Mutations Using Southern Blot and PCR Methodologies Jack Tarleton 1. 3) encoded protein, FMRP, results in mild to moderate mental retardation as its hallmark. Patients with the syndrome often vary dramatically in presentation with a range of intellectual and behavioral deficits, and provide a diagnostic challenge for clinicians due to the subtle nature of the physical phenotype (1,2).

From: Methods in Molecular Biology, vol. 217: Neurogenetics: Methods and Protocols Edited by: N. T. 5 5 15 18 24 25 In general, the number of CGG repeats ranges from 5 – 44 in normal (stable) alleles. In a range of 45 to 54 repeats occasional minor instability may be observed leading to a “gray zone” of borderline alleles. Alleles in the gray zone can be considered normal as women with these alleles have not been observed to have children with fragile X syndrome. Repeats from 55 – 58 have been observed to have more dramatic instability of repeat copy number, yet no direct transmissions to a full mutation have been reported.

3. 10X Bovine serum albumin (BSA): Add 10 mg BSA (Sigma) to 1 mL sterile, deionized H2O. 4. 10X Spermidine (40 mM): Add 152 mg spermidine (Sigma) to 15 mL sterile, deionized H2O. 5. 100 mM Magnesium chloride (MgCl2): Add 203 mg MgCl2 (Sigma) to 1 mL sterile, deionized H2O. 6. 1 g NH4Ac (Sigma) to deionized water. Bring volume to 1 L. Autoclave. 7. 100% ethanol. 8. 8 g Trizma Base (Sigma) to sterile, deionized water. 5. 9. 12 g EDTA (Sigma) to sterile, deionized H2O. Bring volume to 1 L. 10. 5 M EDTA.

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