| Publication Type |
thesis |
| School or College |
School of Medicine |
| Department |
Pathology |
| Author |
Lee, Ming-Chih |
| Title |
Laboratory diagnosis of fragile X syndrome by using rapid cycle polymerase chain reaction |
| Date |
1995-12 |
| Description |
Fragile X syndrome in a common from of inherited mental retardation with an unusual inheritance pattern. The responsible mutation, a large increase in the number of CGG repeats flanking the fragile X mental retardation 1 (FMR-1) gene on the X chromosome, has recently been described. The polymerase chain reaction (PCR) has been used to amplify DNA across the region of CGG repeats to detect the mutation. However, standard procedures take 4-6 hours with low amplification efficiency for long mutated genes that prevent visualization with ethidium bromide staining on agorose gels. In this theses, factors that affect PCR amplification of the FMR-1 region have been systematically studied. These include 1) optimal concentration of MgCl2, dimethyl sulfoxide (DMSO), and 7-deaza-2'deoxyguanosine 5'-triphosphat (7-deaza-dGTP); 2) temperature control of the reaction mixture before the initiation of PCR; 3) denaturation temperature during PCR; and 4) polymerase of polymerase combination used. Optimal concentrations were 1.0 mM Mg2+ (wirh 200 upsilonM of each dNTP), 5-7.5% DNSo, and 0-25% 7-deaza-dGTP substitution of dGTP. A denaturation temperature of 97°C with a rapid temperature transition of the preamplification sample for 0° to 97°C enhanced amplification of high G+C products. Combination of Taq and Pfu polymerase, or KlenTaq1 and Pfu polymerases where Pfu polymerase is used as a minor component with 3' through 5' proofreading activity allow the complete extension of long amplification products from abnormal FMR-1 genes. With systematic optimization, genetic screening of at-risk populations for fragile X with PCR can be improved. |
| Type |
Text |
| Publisher |
University of Utah |
| Subject |
PCR Amplification; Polymerases |
| Subject MESH |
Fragile X Syndrome; Genomics; DNA; Developmental Disabilities |
| Dissertation Institution |
University of Utah |
| Dissertation Name |
MS |
| Language |
eng |
| Relation is Version of |
Digital reproduction of " Laboratory diagnosis of fragile X syndrome by using rapid cycle polymerase chain reaction." Spencer S. Eccles Health Sciences Library. Print version of "Laboratory diagnosis of fragile X syndrome by using rapid cycle polymerase chain reaction." available at J. Willard Marriott Library Special Collection. RJ25.5 1995 .L43 |
| Rights Management |
© Ming-Chih Lee. |
| Format Medium |
application/pdf |
| Format Extent |
2,628,152 bytes |
| Identifier |
undthes,4811 |
| Source |
Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available). |
| Master File Extent |
2,628,214 bytes |
| ARK |
ark:/87278/s6rb76cv |
| Setname |
ir_etd |
| ID |
190872 |
| Reference URL |
https://collections.lib.utah.edu/ark:/87278/s6rb76cv |
