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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林文澧(Win-li Lin) | |
| dc.contributor.author | Chia-Yun Lin | en |
| dc.contributor.author | 林嘉芸 | zh_TW |
| dc.date.accessioned | 2021-06-13T16:42:43Z | - |
| dc.date.available | 2006-07-26 | |
| dc.date.copyright | 2005-07-26 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-06-30 | |
| dc.identifier.citation | 1 Lynch M: Genomics. Gene duplication and evolution. Science 2002; 297: 945-947.
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Acta Neuropathol (Berl) 1996; 92: 454-460. 17 Lopes J, LeGuern E, Gouider R et al: Recombination hot spot in a 3.2-kb region of the Charcot-Marie-Tooth type 1A repeat sequences: new tools for molecular diagnosis of hereditary neuropathy with liability to pressure palsies and of Charcot-Marie-Tooth type 1A. French CMT Collaborative Research Group. Am J Hum Genet 1996; 58: 1223-1230. 18 Yamamoto M, Yasuda T, Mitsuma T, Obara K, Tachi N, Sobue G: [Locations of crossover breakpoints within the CMT 1 A-REP repeat in patients with hereditary neuropathy with liability to pressure palsy--detection by recombinant chromosome-specific polymerase chain reaction]. No To Shinkei 1997; 49: 443-447. 19 Timmerman V, Lofgren A, Le Guern E et al: Molecular genetic analysis of the 17p11.2 region in patients with hereditary neuropathy with liability to pressure palsies (HNPP). Hum Genet 1996; 97: 26-34. 20 MacMillan JC, Upadhyaya M, Harper PS: Charcot-Marie-Tooth disease type 1a (CMT1a): evidence for trisomy of the region p11.2 of chromosome 17 in south Wales families. J Med Genet 1992; 29: 12-13. 21 Ikegami T, Ikeda H, Chance PF et al: Facilitated diagnosis of CMT1A duplication in chromosome 17p11.2-12: analysis with a CMT1A-REP repeat probe and photostimulated luminescence imaging. Hum Mutat 1997; 9: 563-566. 22 Chance PF, Abbas N, Lensch MW et al: Two autosomal dominant neuropathies result from reciprocal DNA duplication/deletion of a region on chromosome 17. Hum Mol Genet 1994; 3: 223-228. 23 Roa BB, Greenberg F, Gunaratne P et al: Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1 neuropathy. Hum Genet 1996; 97: 642-649. 24 Shaffer LG, Kennedy GM, Spikes AS, Lupski JR: Diagnosis of CMT1A duplications and HNPP deletions by interphase FISH: implications for testing in the cytogenetics laboratory. Am J Med Genet 1997; 69: 325-331. 25 Navon R, Timmerman V, Lofgren A et al: Prenatal diagnosis of Charcot-Marie-Tooth disease type 1A (CMT1A) using molecular genetic techniques. Prenat Diagn 1995; 15: 633-640. 26 Latour P, Boutrand L, Levy N et al: Polymorphic short tandem repeats for diagnosis of the Charcot-Marie-Tooth 1A duplication. Clin Chem 2001; 47: 829-837. 27 Badano JL, Inoue K, Katsanis N, Lupski JR: New polymorphic short tandem repeats for PCR-based Charcot-Marie-Tooth disease type 1A duplication diagnosis. Clin Chem 2001; 47: 838-843. 28 Bassler HA, Flood SJ, Livak KJ, Marmaro J, Knorr R, Batt CA: Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl Environ Microbiol 1995; 61: 3724-3728. 29 Wilke K, Duman B, Horst J: Diagnosis of haploidy and triploidy based on measurement of gene copy number by real-time PCR. Hum Mutat 2000; 16: 431-436. 30 Aarskog NK, Vedeler CA: Real-time quantitative polymerase chain reaction. A new method that detects both the peripheral myelin protein 22 duplication in Charcot-Marie-Tooth type 1A disease and the peripheral myelin protein 22 deletion in hereditary neuropathy with liability to pressure palsies. Hum Genet 2000; 107: 494-498. 31 Thiel CT, Kraus C, Rauch A, Ekici AB, Rautenstrauss B, Reis A: A new quantitative PCR multiplex assay for rapid analysis of chromosome 17p11.2-12 duplications and deletions leading to HMSN/HNPP. Eur J Hum Genet 2003; 11: 170-178. 32 Noonan KE, Beck C, Holzmayer TA et al: Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc Natl Acad Sci U S A 1990; 87: 7160-7164. 33 Celi FS, Zenilman ME, Shuldiner AR: A rapid and versatile method to synthesize internal standards for competitive PCR. Nucleic Acids Res 1993; 21: 1047. 34 Nelis E, Haites N, Van Broeckhoven C: Mutations in the peripheral myelin genes and associated genes in inherited peripheral neuropathies. Hum Mutat 1999; 13: 11-28. 35 Takashima H, Boerkoel CF, Lupski JR: Screening for mutations in a genetically heterogeneous disorder: DHPLC versus DNA sequence for mutation detection in multiple genes causing Charcot-Marie-Tooth neuropathy. Genet Med 2001; 3: 335-342. 36 Choy YS, Dabora SL, Hall F et al: Superiority of denaturing high performance liquid chromatography over single-stranded conformation and conformation-sensitive gel electrophoresis for mutation detection in TSC2. Ann Hum Genet 1999; 63 ( Pt 5): 383-391. 37 Buyse IM, Fang P, Hoon KT, Amir RE, Zoghbi HY, Roa BB: Diagnostic testing for Rett syndrome by DHPLC and direct sequencing analysis of the MECP2 gene: identification of several novel mutations and polymorphisms. Am J Hum Genet 2000; 67: 1428-1436. 38 Pan CL, Tseng TJ, Lin YH, Chiang MC, Lin WM, Hsieh ST: Cutaneous innervation in Guillain-Barre syndrome: pathology and clinical correlations. Brain 2003; 126: 386-397. 39 Shi HN, He RG, Zhou XJ et al: Hereditary Neuropathy with Liability to Pressure Palsies: A Clinical and Electrophysiological Study. Acta Neurol Taiwan 1997; 6: 191-196 40 Stronach EA, Clark C, Bell C et al: Novel PCR-based diagnostic tools for Charcot-Marie-Tooth type 1A and hereditary neuropathy with liability to pressure palsies. J Peripher Nerv Syst 1999; 4: 117-122. 41 Yau SC, Bobrow M, Mathew CG, Abbs SJ: Accurate diagnosis of carriers of deletions and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage analysis. J Med Genet 1996; 33: 550-558. 42 Zhu D, Kennerson M, Merory J et al: Refined localization of dominant intermediate Charcot-Marie-Tooth neuropathy and exclusion of seven known candidate genes in the region. Neurogenetics 2003; 4: 179-183. 43 Kim SW, Lee KS, Jin HS et al: Rapid detection of duplication/deletion of the PMP22 gene in patients with Charcot-Marie-Tooth disease Type 1A and hereditary neuropathy with liability to pressure palsy by real-time quantitative PCR using SYBR Green I dye. J Korean Med Sci 2003; 18: 727-732. 44 Rowland JS, Barton DE, Taylor GR: A comparison of methods for gene dosage analysis in HMSN type 1. J Med Genet 2001; 38: 90-95. 45 Akrami SM, Rowland JS, Taylor GR, Armour JA: Diagnosis of gene dosage alterations at the PMP22 gene using MAPH. J Med Genet 2003; 40: e123. 46 Frisso G, Carsana A, Tinto N, Calcagno G, Salvatore F, Sacchetti L: Direct detection of exon deletions/duplications in female carriers of and male patients with Duchenne/Becker muscular dystrophy. Clin Chem 2004; 50: 1435-1438. 47 Poropat RA, Nicholson GA: Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR. Clin Chem 1998; 44: 724-730. 48 Bernard R, Boyer A, Negre P et al: Prenatal detection of the 17p11.2 duplication in Charcot-Marie-Tooth disease type 1A: necessity of a multidisciplinary approach for heterogeneous disorders. Eur J Hum Genet 2002; 10: 297-302. 49 Young P, Stogbauer F, Wiebusch H et al: PCR-based strategy for the diagnosis of hereditary neuropathy with liability to pressure palsies and Charcot-Marie-Tooth disease type 1A. Neurology 1998; 50: 760-763. 50 Beckmann A, Schroder JM: Screening for Charcot-Marie-Tooth type 1A and hereditary neuropathy with liability to pressure palsy in archival nerve biopsy samples by direct-double-differential PCR. Acta Neuropathol (Berl) 2000; 100: 459-463. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38703 | - |
| dc.description.abstract | 研究已知有許多遺傳疾病是由特定基因點突變所引起，近來發現愈來愈多的基因疾病是由於基因拷貝數的改變所造成。而 Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) 疾病發生的原因即由於位在染色體 17p11.2-12 上之1.5-Mb區域重複或缺失所造成。由於此區域內含有一周邊髓磷蛋白(Peripheral myelin protein-PMP22)基因，此區域若發生重複或缺失就會造成此兩種不同的疾病產生。
本論文所提出的方法是利用多重聚合酶連鎖反應(multiplex PCR)策略，同時放大一未知基因拷貝數的待測區域和另一已知基因拷貝數之參考區域，之後藉由DNA片段突變分析儀 (DHPLC) 或毛細管電泳 (CE) 檢測出基因拷貝數的微量差異。在此研究中，我們對總共 110個已經診斷的個體，包括已確診的32位CMT1A病人，17位HNPP病人及61位正常人進行分析，實驗使用相同多重聚合酶連鎖反應的流程，結果顯示，無論是使用DNA片段突變分析儀或是毛細管電泳，兩個系統所定量出來的PMP22基因拷貝數相符合，並且所有樣品均使用聚合酶連鎖反應-DNA限制酶片段長度多型性(PCR-RFLP)分析確認結果。 在此篇論文中，我們證明結合多重聚合酶連鎖反應與DNA片段突變分析儀的分析方法在檢測PMP22基因拷貝數發生重複或缺失所造成CMT1A或HNPP疾病時，是一種高效率、高準確率、高可信度的技術，並且除了可以定量基因拷貝數，還可以鑑定一般常見已知的突變基因型以及不同族群之新的突變點位，故此技術未來應可成為臨床基因檢測之一項利器。 | zh_TW |
| dc.description.abstract | Many genetic diseases are caused by the presence of point mutations in respective genes; an increasing number of diseases are known to be caused by changes in gene copy number. Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are caused by a 1.5 Mb duplication and deletion at chromosome 17p11.2-12 encompassing the peripheral myelin protein (PMP22).
We developed a rapid and reliable detection system for duplications/deletions of PMP22 gene based on measurement of gene copy number. The method involves amplifications of a test locus with unknown copy number and a reference locus with known copy number by multiplex PCR strategy, following by Denaturing high-performance liquid chromatography (DHPLC) or capillary electrophoresis detection to identify single copy changes. Thirty-two patients with CMT1A, seventeen patients with HNPP, and sixty-one unaffected individuals were analyzed. By using the same multiplex competitive PCR protocol, the measured PMP22 gene dosage revealed the compatible results in DHPLC and capillary electrophoresis analysis. The results of multiplex PCR/DHPLC or multiplex PCR/capillary electrophoresis assay were all confirmed by PCR-RFLP analysis. We demonstrated that multiplex PCR/DHPLC assay is an efficient, accurate, and reliable technique for the gene dosage determination of PMP22 gene for CMT1A duplication and HNPP deletion. It also can detect the common known mutations and the novel mutations in varied population. Hence the technique could be a power tool in clinical genetic screening. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T16:42:43Z (GMT). No. of bitstreams: 1 ntu-94-R92548018-1.pdf: 1435603 bytes, checksum: 2da6717729fabc8cee401fb7b3187823 (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | 圖目錄.………………………………………….…..….…… VII
表目錄……………………………….………….…..……… VIII 中文摘要.…………………………………………………......IX 英文摘要………………………………………………….…...X 一、緒論……………………………………………………….……1 1-1簡介………………………………………………………………..1 1-2分子研究歷史簡述………………………………………………..2 1-2-1 基因序列重複片段的發現……………………………………..2 1-2-2 PMP22基因是CMT1A的致病基………………………………..3 1-2-3 重組熱點的研究………………………………………………..4 1-3診斷方法與臨床應用……………………..……………………..4 1-4聚合酶連鎖反應之原理…………………..……………………..7 1-5多重聚合酶連鎖反應之原理…….………..……………………..8 1-6毛細管電泳(CE)之原理…….………..…………………………..9 1-7DNA片段突變分析儀(DHPLC)之原理………………………...10 1-7-1 DNA 片段突變分析儀之裝置…………………………………11 1-8研究動機與方向………………………………................12 二、實驗材料與方法………………………………….............14 2-1 實驗材料……………………………………................14 2-1-1 人類基因體 DNA (Human Genomic DNA) …..............14 2-1-2 引子……………………………………...................14 2-1-3 其他反應試劑…….....................14 2-2 實驗儀器………………………….........................15 2-3 實驗方法…………………………………...................16 2-3-1 抽取DNA………………………………....................16 2-3-2 聚合酶連鎖反應-DNA限制酶片段長度多型性 (PCR-RFLP)...............16 2-3-3 多重聚合酶連鎖反應 (Multiplex Polymerase Chain Reaction，PCR).....16 2-3-4 單點突變之聚合酶連鎖反應 (PCR for Single Nucleotide Mutations).....17 2-3-5 洋菜凝膠電泳 (Agarose gel electrophoresis) ......................................17 2-3-6 毛細管電泳分析儀(CE) 與 DNA片段突變分析儀(DHPLC) ………...18 2-3-7 資料分析………………………………................18 2-3-8 DNA片段突變分析儀分析單點突變(DHPLC Analysis for Single Nucleotide Mutations) …………………............19 三、結果…...…………………………………………………………20 3-1 聚合酶連鎖反應-DNA限制酶片段長度多型性 (PCR-RFLP).…20 3-2 聚合酶連鎖反應 (PCR) 結果……………………………………20 3-3 毛細管電泳 (CE) ……………….…...…………………….…21 3-4 DNA片段突變分析儀 (DHPLC) .……………..……..…….…21 3-5 資料分析.….….………………….…………………………….22 3-6 DHPLC突變分析檢測……………….…………………………….23 四、討論…..…………………………………………………………24 五、結論……...………………………………………………………27 5-1 總結………………………………………………………………27 5-2未來展望…………………………………………………..28 六、參考文獻………………………………………………….…...29 圖目錄 圖一 PMP22基因產物之結構- 4個公認的跨膜區域…………..…………….………34 圖二 不平衡交換造成CMT1A/HNPP的機制……………………………….………..35 圖三 即時定量PCR(Real-time PCR)之簡單原理示意圖…………………….……….36 圖四 聚合酶連鎖反應之簡單原理示意圖……………………………………..………37 圖五 洋菜膠電泳確認RFLP結果……………………………………….…….……….38 圖六 多重聚合酶連鎖反應結果以洋菜膠電泳確認…………………….……….…....39 圖七 毛細管電泳之分析圖譜………………………………………….….……..……..40 圖八 不同循環參數的DNA片段突變分析儀之分析圖譜……………….……….…..41 圖九 不同條件下的多重聚合酶連鎖反應之DNA片段突變分析儀分析圖譜……....42 圖十 毛細管電泳之數據統計圖…………………………………..................................43 圖十一 DNA片段突變分析儀的數據統計圖………………………………….……...44 圖十二 利用DNA片段突變分析儀與毛細管電泳所量測計算値的相關圖….….…..45 圖十三 DNA片段突變分析儀的數據統計圖-PMP22 基因外顯子2……….……...46 圖十四 本研究所發現之MPZ基因致病突變點位………………………………......47 表目錄 表一 RFLP所需之Long-Range PCR之引子對…………………………….….…….48 表二 RFLP所需之Long-Range PCR之各反應試劑的濃度與總體積………….…..49 表三 RFLP所需之Long-Range PCR之PCR熱循環參數……………………….…50 表四 多重聚合酶連鎖反應所需的引子對………………………………………..…..51 表五 多重聚合酶連鎖反應試劑的濃度與總體積-1…………………..………….….52 表六 多重聚合酶連鎖反應之PCR熱循環參數……………………...…….….……..53 表七 多重聚合酶連鎖反應之反應試劑的濃度與總體積-2………...…….……..…..54 表八 單點突變分析之聚合酶連鎖反應所需的引子對以及DHPLC之沖提條件…………………...…………………………………………..……………55、56 表九 單點突變分析之聚合酶連鎖反應-反應試劑的濃度、總體積與繞循環參數.………………………………………………………………………..……….57 表十 多重聚合酶連鎖反應之DHPLC各緩衝溶液之成分及沖提條件…….…...….58 表十一 多重聚合酶連鎖反應量測PMP22 外顯子2之基因拷貝數………..………59 表十二 利用DNA突變分析儀以及毛細管電泳所量測PMP22基因拷貝數之 數值統計總表…………………………………...……………………..……...60 表十三 DHPLC所檢測PMP22, MPZ and GJB1基因內之單點突變統整表……..…61 | |
| dc.language.iso | zh-TW | |
| dc.subject | 進行性腓骨肌萎縮症 | zh_TW |
| dc.subject | DNA突變分析儀 | zh_TW |
| dc.subject | 基因劑量 | zh_TW |
| dc.subject | 周邊神經髓鞘蛋白基因 | zh_TW |
| dc.subject | 遺傳性壓力敏感性周圍神經病 | zh_TW |
| dc.subject | 毛細管電泳 | zh_TW |
| dc.subject | CMT1A | en |
| dc.subject | gene dosage | en |
| dc.subject | PMP22 gene | en |
| dc.subject | HNPP | en |
| dc.subject | capillary electrophoresis | en |
| dc.subject | Denaturing high-performance liquid chromatography (DHPLC) | en |
| dc.title | 建立藉由多重聚合酶連鎖反應結合 DNA 突變分析儀檢測 CMT1A/HNPP疾病致病因子–PMP22 基因之快速且可信的基因診斷系統 | zh_TW |
| dc.title | A Rapid and Reliable Detection System for the Analysis of CMT1A/HNPP disease causing factor -PMP22 Gene by Multiplex PCR/DHPLC Assay | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 蘇怡寧(Yi-ning Su) | |
| dc.contributor.oralexamcommittee | 李鴻(Hung Li),謝松蒼(Sung -Tsang Hsieh),陳祈安(Chi-An Chen) | |
| dc.subject.keyword | DNA突變分析儀,毛細管電泳,進行性腓骨肌萎縮症,遺傳性壓力敏感性周圍神經病,周邊神經髓鞘蛋白基因,基因劑量, | zh_TW |
| dc.subject.keyword | Denaturing high-performance liquid chromatography (DHPLC),capillary electrophoresis,CMT1A,HNPP,PMP22 gene,gene dosage, | en |
| dc.relation.page | 61 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-07-01 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| 顯示於系所單位： | 醫學工程學研究所 | |
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