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研究生:白崇彥
研究生(外文):Chung-yen Pai
論文名稱:刑事標誌系統在口腔細胞檢體之DNA變異分析與穩定性評估
論文名稱(外文):DNA Alteration Analysis and Stability Evaluation of Forensic Marker Systems in Buccal Cell Samples
指導教授:徐邦達徐邦達引用關係
指導教授(外文):Ban-dar Hsu
學位類別:博士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:107
中文關鍵詞:短重覆序列DNA變異失去異質性嚼食檳榔口腔細胞
外文關鍵詞:STRallelic alterationLOHBQ-chewer
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摘要
嚼食後的檳榔渣存留有咀嚼者的口腔上皮脫落細胞,因此乃刑案現場之重要跡證。口腔上皮細胞為刑事檢驗及親子鑑定常用之檢體,醫用之口腔切片組織也被用在空難後之人別身分辨識與確認。但眾所週知的是嚼食檳榔會造成口腔癌及相關的口腔疾病,且腫瘤組織的基因組DNA序列常出現突變或不穩定的現象,究竟刑事所用的DNA標誌系統在口腔癌組織的穩定性如何?會產生哪些變異型態?而在未罹患口腔癌之嚼食檳榔者的口腔細胞是否有可能產生前期性的口腔傷害,而致使這些刑事鑑定所用的多種基因座的基因穩定性受到挑戰。此一穩定性的調查與確認,直接關係著人別鑑識結果是否誤判,因此就刑事應用而言,是非常有必要,也是本研究的主要目的之一。另一方面,從嚼食後的檳榔渣無法萃取出咀嚼者的DNA,因此本研究的另一個目的為發展出一套有效的萃取方法,以解決實務警察機關所面臨的困難。
受測者分為三群,口腔癌患者100人,嚼食檳榔者100人,無嚼食檳榔習慣者100人(作為控制組),合計300人。蒐集其口腔細胞與血液細胞,萃取DNA,以商業化之鑑驗盒配合DNA 自動定序儀與其分析軟體來比對同一人的口腔與血液細胞內的基因標誌,最後再以統計(ANOVA, x2, Scheffe test)分析計算、比較所有出現變異之基因座。所分析的基因座均為現今刑事實驗室常用的基因標誌,主要有兩種商業化之鑑驗盒,其一為AmpliType HLA-DQA1/PM multiplex system,包含HLA-DQA1及PM(LDLR,GYPA,HBGG,D7S8,GC)合計6個基因。另 一為AmpFlSTRTM multiplex system, 包含9個STR基因座(D3S1358、vWA、FGA、TPOX、TH01、CSF1PO、D5S818、D13S317、D7S812)與一個性別基因。而在檳榔渣之人類DNA的萃取方法研發方面,則先比較分析三種傳統方法(salt/chloroform, 5% chelex-100 resin, QIAamp)萃取存放八週之模擬檢體所得到的結果,並根據其分別的萃取原理找出失敗的可能原因,再以化學藥劑(PVP與CTAB)結合抑制檳榔渣中的多酚類與多醣類物質之策略來研發萃取方法。
在口腔癌檢體方面,共發現兩種DNA變異型態,其一為major allelic imbalance;另一為allelic alterations (又分為expansion, contraction, un-classified三種亞型)。整體不穩定率為33%,兩種變異型態之嚴重度均與病理期成正比例。長度多型之標誌總共發生44個major allelic imbalance與25個allelic alterations,序列多型之標誌僅發現一個基因不吻合。而在嚼食檳榔者之檢體方面,長度多型之標誌發現兩個基因座出現major allelic imbalance,但仍能清楚辨識為異質型組合;序列多型標誌則全部吻合。因此,對於口腔癌檢體而言,序列多型標誌比長度多型標誌穩定;而對於嚼食檳榔者之檢體而言,兩種系統均可使用。統計分析結果則顯示:口腔癌檢體之九個STR基因座的allelic imbalance平均值與其對應的控制組有顯著差異 (p value 均小於0.05 ),但基因座彼此之間則無發生率之顯著差異(p value = 0.19);口腔癌患者中之嚼食檳榔因素方面,嚼食者在兩種變異型態均比未嚼食者出現顯著差異之高發生率,但癌組織所發生的變異型態種類則不受嚼食檳榔因素所影響(p value 分別為0.15與0.57)。另一方面,所研發的”PVP/CTAB萃取法” 對於陳舊之檳榔模擬檢體與存放四年之50個刑案檳榔檢體,其成功率分別為100%與92%,因此本法對於咀嚼後檳榔渣人類DNA之萃取確實有效。
Abstract
Chewed betel-quid (BQ) residues are often considered vital biological evidence at crime scenes, since the human DNA extracted from the residues is actually from buccal epithelial cells and can be associated with suspects. However, BQ-chewing is also a risk factor for oral diseases and/or cancers. Although archived medical oral-specimens can be used to identify specific individuals under adverse conditions, STR markers are known to be unstable in various tumor tissues. On the other hand, the forensic analysis of BQ evidence has been hindered by failures in extraction of human DNA for PCR analysis. Therefore, it is a prerequisite for relevant forensic casework to establish a reliable method for extracting DNA from chewed BQ residues.
The stability of forensic marker systems of both sequence and length polymorphism was first investigated and then used to evaluate the forensic appropriateness of the oral samples of both healthy betel quid (BQ) chewers and the archived clinical specimens from oral cancer patients.
The analyses were performed on buccal samples from 100 BQ-chewers and 100 oral cancer patients, and corresponding peripheral blood samples from the same subject. A group of 100 non-BQ-chewers were used as the control. The genotypes of oral and paired peripheral blood samples were compared, using the commercialized typing systems of HLA-DQA1, PM (including LDLR, GYPA, HBGG, D7S8, and GC loci), and AmpFlSTRTM markers (including 9 STR loci and the Amelogenin gene). As for the development of human DNA extracted from chewed BQ-residues, three conventional methods (salt/chloroform, 5 % Chelex-100 resin, and QIAamp) were first tested for extraction of human DNA from 33 mock BQ samples, which had been stored for less than two months, and 50 four-year old forensic BQ samples. PCR amplifications from the HLA-DQA1&PM and the STR loci were then used to test the quality of the extracted DNA.
In the group of 100 oral cancer patients, two types of DNA instability were found. They were major allelic imbalances, and allelic alterations including expansion, contraction and the un-classified types (can not be confirmed as the expansion or the contraction). The overall percentage of the cancerous subjects demonstrating DNA instability was 33% (5 patients possessing both types of DNA instability). Both types of DNA instability showed a tendency of increasing with the severity of the pathological stage of oral cancer. Forty-four occurrences of major allelic imbalance were found in 21 cancer patients. The statistical result revealed that there was no significant difference in the allelic imbalanced occurrence among the nine STR loci. Allelic alterations were found in 17 patients, within which 12 individuals had the expansion, 5 had the contraction, and 3 had the un-classified type. Further, among these 17 patients, 3 were found to acquire multiple allelic alterations at multiple loci. For the DNA marker of sequence polymorphism, one discordant result at D7S8 was found in the 600 DQA1/PM-marker loci. The 100 healthy BQ-chewers had consistent oral swab and paired blood sample genotypes analyzed with both DQA1/PM and STR marker systems, although two loci with major allelic imbalance were detected. However, the two imbalanced alleles were virtually half lost, and could still be recognized as heterozygous alleles. The statistical results of ANOVA, x2, and Scheffe tests indicated that the means of allelic imbalance at the 9 STR loci of the oral cancerous group revealed a significant difference from those in the control group.
The findings herein suggest that when cancerous specimens were tested, the HLA-DQA1/PM system with point polymorphism appears more reliable than the STR system with length polymorphism. Our results also indicate that healthy BQ-chewers’ oral cotton swabs containing buccal epithelial cells are useful for forensic purposes using the HLA-DQA1, PM, and STR marker systems.
In order to solve the problem in PCR analysis of DNA from old BQ samples, we developed a DNA extraction method based on the use of polyvinyl pyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB), which bind to two common classes of PCR inhibitors in plants, polyphenols and polysaccharides, respectively. The result showed that this “PVP/CTAB” method is completely successful for the mock BQ samples, and 92% (46 out of 50) successful for the four-year old forensic BQ samples. To our best knowledge, this is the first report of a reliable method for the extraction of human DNA for PCR from chewed BQ residues. This method should provide a useful means for forensic identification in countries where betel-chewing is common.
DNA Alteration Analysis and Stability Evaluation of Forensic Marker Systems in Buccal Cell Samples
Chinese abstract…………………………………………………………3
English abstract………………………………………………………… 5
Chapter 1 DNA alterations at the STR loci in samples of oral cancer tissue and betel quid chewers’ oral epithelial cells ………. 7
Introduction…………………………………………………………..7
Materials and methods………………………………………………10
Results……………………………………………………………… 15
Discussion………………………………………………………….. 19
Summary…………………………………………………………… 31
References………………………………………………………….. 32
Chapter 2 DNA stability in HLA-DQA1/PM markers and genetic instability in STR markers: implications for forensic application………………………………………………...35
Introduction………………………………………………………….35
Materials and methods…………………………………………….…37
Results and discussion……………………………………………….40
Summary…………………………………………………………… 49
References………………………………………………………….. 50
Chapter 3 Extraction of human DNA for PCR from chewed residues
of betel quid using a novel“PVP/CTAB”method…………53
Introduction………………………………………………………..53
Materials and methods…………………………………………….55
Results and discussion……………………………………………..61
Summary…………………………………………………………..69
References………………………………………………………...70
Appendix
Part A Association analyses of monoamine oxidase (MAO) genes and sexually aggressive behavior ………………………72
Introduction………………………………………………………..72
Materials and methods……………………………………………..75
Results……………………………………………………..………79
Discussion…………………………………………………………..82
Summary……………………………………………………………93
References…………………………………………………………. 94
Part B Case-control study of catechol O-methyltransferase (COMT) polymorphisms in sexually aggressive behavior…………97
Introduction…………………………………………………………97
Materials and methods………………………………………………99
Results and discussion……………………………………………. 101
Summary…………………………………………………………..106
References…………………………………………………………107
Reference:
1 A.J. Jeffreys, M.J. Jeffreys, E. Hagelberg, A. Sonnberg, Identification of the skeletal remains of Josef Mengele by DNA analysis, Forensic Sci. Int. 56 (1992) 65-76.
2 C.P. Kimpton, D. Fisher, S. Watson, M. Adams, A. Urquhart, J. Lygo, P. Gill, Evaluation of an automated DNA profiling system employing multiplex amplification of four tetrameric STR loci, Int. J. Legal Med.106 (1994) 302-311.
3 J.E. Lygo, P.E. Johnson, D.J. Holdway, S. Woodroffe, J.P. Whitaker, T.M. Clayton, C.P. Kimpton, P. Gill, The validation of short tandem repeat (STR) loci for use in forensic casework, Int. J. Legal Med.107 (1994) 77-89.
4 P. Gill, P.I. Ivanov, C. Kimpton, R. Piercy, N. Benson, G. Tully, I. Evett, E. Hagelberg,K.M. Sullivan, Identification of the remains of the Romanov family by DNA analysis, Nature Genet. 6 (1994) 130-135.
5 E. Hagelberg, I.C. Gray, A.J. Jeffreys, Identification of the skeletal remains of a murder victim by DNA analysis, Nature. 352 (1991) 427-429.
6 A.J. Jeffreys, M.J. Allen, E. Hagelberg, A. Sonnberg, Identification of skeletal remains of Josef Mengele by DNA analysis, Forensic Sci. Int. 56 (1992) 65-76.
7 J.L. Weber, C. Wong, Mutation of human short tandem repeats, Hum. Mol. Genet. 2 (1993) 1123-1128.
8 M. Mitas, Trinucleotide repeats associated with human disease, Nucleic Acids Res. 25 (1997) 2245-2253.
9 M. Alwazzan, E. Newman, M.G. Hamshere, J.D. Brook, Myotonic dystrophy is associated with a reduced level of RNA from the DMWD allele adjacent to the expanded repeat, Human Mol. Genet. 8 (1999) 1491-1497.
10 K.C. Halling, J. Harper, C.A. Moskaluk, S.N. Thibodeau, G. R. Petroni, A.S. Yustein, P. Tosi, C. Minacci, F. Roviello, P. Piva, S.R. Hamilton, C.E. Jackson, S.M. Powell, Origin of microsatellite in gastric cancer, Am. J. Pathol. 155 (1999) 205-211.
11 F.C. Schmitt, R. Soares, H. Gobbi, F. Milanezzi, F. S. Santos, L. Cirnes, C. Costa, R. Seruca, Microsatellite instability in medullary breast carcinomas, Int. J. Cancer 82 (1999) 644-647.
12 C. Yeun-Jun, S. Ji-Min, L. Joo-Young, J. Yong-Tae, S. Eun-Joo, C. Sang-Wook R. Mun-Gan, Microsatellite instability-associated mutations associate preferentially with the instestinal type of primary gastric carcinomas in a high-risk population, cancer research 56 (1996) 4662-4665.
13 R.T. Burks, T.D. Kessis, K.R. Cho, L. Hedrick, Microsatellite instability in endometrial carcinoma, Oncogene 9 (1994) 1163-1166.
14 E. Rizos, G. Sourvinos, D.A. Spandidos, Loss of heterozygosity at 8p, 9p, and 17q in laryngeal cytological specimens, Oral Oncol. 34 (1998) 519-523.
15 M. Velickovic, B. Delahunt, S.K. Grebe, Loss of heterozygosity at 13p14.2 in clear cell renal cell carcinoma is an early event and is highly localized to the FHIT gene locus, Cancer Res. 59 (1999) 1323-1326.
16 R.F. Schwerdtle, S. Storkel, C. Neuhaus, H. Brauch, E. Weidt, W. Brenner, Allelic losses at chromosomes 1p, 2p, 6p,10p,13q,17p, and 21q significantly correlate with the chromophobe subtype of renal cell carcinomas, Cancer Res. 56 (1996) 2927-2930.
17 P. Hoff-Olsen, S. Jacobsen, B. Mevag, B. Olaisen, Microsatellite stability in human post-mortem tissues, Forensic Sci. Int. 119 (2001) 273-278.
18 C.M. Hsu, N.E. Huang, L.C. Tsai, C.H. Chao, A. Linacre, J.C. Lee, Identification of victims of the 1998 Taoyuan Airbus crash accidenct using DNA analysis, Int. J. Legal Med. 113 (1999) 43-46.
19 J.P. Whitaker, T.M. Clayton, A.J. Urquhart, E.S. Kimpton, P. Gill, Short tandem repeat typing of bodies from a mass disaster: high success rate and characteristic amplification patterns in highly degraded samples, Biotechniques 18 (1995) 670-677.
20 R.J. Rubocki, K.J. Duffy, K.L. Shepard, B.J. McCue, S.J. Shepherd, J.L. Wisecarver, Loss of heterozygosity detected in a short tandem repeat (STR) locus commonly used for human DNA identification, J. Forensic Sci. 45 (2000) 1087-1089.
21 Y.C. Ko, Y.L. Huang, C.H. Lee, M.J. Chen, L.M. Lin, C.C. Tsai, Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan, J. Oral Pathol. Med. 24 (1995) 250-453.
22 J.H. Jeng, L.J. Hahn, B.R. Lin, C.C. Hsieh, C.P. Chan, M.C. Chang, Effects of areca nut, inflorescence piper betle extracts and arecoline on cytotoxicity, total and unscheduled DNA synthesis in cultured gingival keratinocytes, J. Oral Pathol. Med. 28 (1999) 64-71.
23 F.S. Chiou, C.Y. Pai, Y.P.P. Hsu, C.W. Tsai, C.H. Yang, Extraction of human DNA for PCR from chewed residues of betel quid using a novel “PVP/CTAB” method, J. Forensic Sci. 46 (2001) 166-177.
24 R. Mullenbach, P.J.L. Lagoda, C. Welter, An efficient salt chloroform extraction of DNA from blood and tissues, Trends Genet. 5 (1989) 391.
25 J.C. Boyer, A. Umar, J.I. Risinger, J.R. Lipford, M. Kane, S. Yin, J. C. Barrett, R.D. Kolodner, T.A. Kunkel, Microsatellite instability, mismatch repair deficiency, and genetic defects in human cancer cell lines, Cancer Res. 55 (1995) 6063-6070.
26 B. Brinkmann, M. Klintschar, F. Neuhuber, J. Huhne, B. Rolf, Mutation rate in human microsatellites: influence of the structure and length of the tandem repeat, Am. J. Hum. Genet. 62 (1998) 1408-1415.
27 Hochemister MN, Budowle B, Jung J, Borer UV, Comey CT, Dirnhofer R et al. PCR-based typing of DNA extracted from cigarette butts. Int. J. Leg. Med.1991; 104: 224-33.
28 Hopkins B, Williams NJ, Webb MBT, Debenham PG, Jeffery AJ. The use of minisatlite variant repeat-polymerase Chain Reaction(MVR-PCR) to determine the source of saliva on a used postaged stamp. J. Forensic Sci.1994;39: 526-31.
29 Sweet DJ, Identification de manchas de saliva de humana mediante el enalisis DNA. Doctoral Thesis, Univ. of Granda. 1995.
30 Wang SM, Ling YC, Tsai LC, Giang YS. Headspace sampling and gas chromatographic-mass spectrometric determination of amphetamine and methamphetamine in betel. J Chromatogr. 1989; 475: 447
31 Ko YC, Chang SJ, Hesieh SF. Prevalence of betel quid chewing habit in Taiwan and related sociademographic factors. J. Oral Pathol. Med. 1992;21:261-64.
32 IRAC. Betel-Quid and area nut chewing. Lyon: International Agency for Research on Cancer. Monographs. 1986;37:141-202
33 Walsh DJ, Coreu AC, Cotton RW, Forman L, Herrin GL, Word CJ, et al. Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva. J of Forensic Sci 1992; 37: 387-95
34 Sweet D, Lorente M, Lorente JA. Valenzuela A, Villanueva E. An improved method to recover saliva from human skin: the double swabs techniques. J Forensic Sci 1997;42:320-22
35 Fridez F, Coquoz R. PCR DNA typing of stamps: evaluation of the DNA extraction. Forensic Sci Int 1996;78:103-10
36 Mullenbach R, Lagoda PJL, Welter C. An efficient salt chloroform extraction of DNA from blood and tissues. Trends Genet 1989; 5:391
37 Walsh PS, Metzger DA, Higuchi R. ChelexR 100 as a mediumn for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 1991;10:506-13
38 Waye JS, Presley LA, Budowle B, Shutler GG, Fourney RM. A simple and sensitive method for quantifying human genomic DNA in forensic specimen extracts. BioTechniques 1989;7:852-55
39 Gill P, Sparkes R, Kimpton C. Development of guidelines to designate alleles using an STR multiplex system. Forensic Sci Int 1997;89:185-97
40 Koonul K P, Brandt W F, Lindesy G, Farrant JM. Inhibitory effect of polyphenolic during PCR. 2nd International Electronic Conference on Synthetic Organic Chemistry (ECSOC-2). 1998: 1-30
41 Scott OR, Bendich A J. Extraction of total DNA from plants, Algae and Fungi Plant Mol. Bio. Manual 1994:D1:1-8
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