Retrospective Fragile X Study by Capillary and Agarose Gel Electrophoresis

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Publication Type thesis
School or College School of Medicine
Department Pathology
Author Baker, Christine Lee
Title Retrospective Fragile X Study by Capillary and Agarose Gel Electrophoresis
Date 2011-05
Description Fragile X Syndrome (FxS) is the most common cause of inherited mental disability and autism spectrum disorder. Estimated incidence in males is 1 in 4000 and 1 in 7000 for females. The gene is on the distal end of the long arm of the X chromosome and contains a trinucleotide cytosine, guanine, guanine (CGG) microsatellite repeat in the 5' untranslated region (5'UTR) of the gene. When the repeat region is fully expanded the CpG island near the gene promoter site becomes methylated, and the result is silencing of the gene and loss of protein product. Health issues that correspond to a partially expanded trinucleotide repeat are Fragile X Tremor and Ataxia Syndrome (FXTAS) and Primary Ovarian Insufficiency (POI). Current molecular detection involves Polymerase Chain Reaction (PCR) amplification of the CGG region, followed by capillary electrophoresis (CE) separation and fluorescent detection of the PCR amplicon with very high sizing resolution for normal and lower range premutation repeats. For larger pre-mutation repeats and full mutations, detection is done with digestion of genomic DNA with methylation sensitive enzymes, followed by Southern blotting. The Southern blots have some significant disadvantages; they are labor intensive, require large amounts of genomic DNA, blot sizing of the CCG repeat region has very imprecise resolution, reagents are expensive and they have a long processing time. Fluorescent detection of PCR product by CE is faster, less expensive, and has iv much higher resolution, but it has not been useful for the detection of full mutations due to PCR inefficiency for large CGG repeats and difficulty of detecting a very large CG rich trinucleotide repeat PCR product. The purpose of this thesis is an overview of the current molecular paradigms of FxS, FXTAS and POI, molecular testing and a comprehensive evaluation of the Celera Fragile X assay and the possibility that use of their 1.5% agarose recipe, gel electrophoresis (GE) and a long injection capillary electrophoresis protocol can detect all premutation and full mutation samples and reduce the need for Southern blotting for molecular detection of full mutation patients.
Type Text
Publisher University of Utah
Subject MESH Fragile X Syndrome; Electrophoresis, Agar Gel; Electrophoresis, Capillary; Polymerase Chain Reaction; Blotting, Southern; Fragile X Mental Retardation Protein; Sensitivity and Specificity; Diagnostic Techniques and Procedures
Dissertation Institution University of Utah
Dissertation Name Master of Science
Language eng
Relation is Version of Digital reproduction of Retrospective Fragile X Study by Capillary and Agarose Gel Electrophoresis. Spencer S. Eccles Health Sciences Library. Print version available at J. Willard Marriott Library Special Collections.
Rights Management Copyright © Christine Lee Baker 2011
Format Medium application/pdf
Format Extent 762,982 bytes
Source Original in Marriott Library Special Collections, RJ25.5 2011.B34
ARK ark:/87278/s6dg00df
Setname ir_etd
Date Created 2014-04-10
Date Modified 2017-05-11
ID 196279
Reference URL
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