臺灣博碩士論文加值系統

摘 要
分子標記技術廣泛應用於遺傳研究的絕佳分析工具，高度多形性和再現性
微衛星分子標記遍佈高等生物基因組；傳統型核酸長度標準物涵蓋密度不足，
致使判讀數據為相對移動距離比值的非整數相對數值，無法準確分析核苷酸自
然長度的正整數絕對數值，因而數據整合不易甚至分析推論結果失誤。本研究
目標建立高密度絕對長度核酸標準物MarkQoff，配合自動影像數據分析Aida
軟體，完整技術平台任務目標為絕對長度數值的電泳條帶判讀結果，助益相容
整合多方實驗數據與提升精確數據分析結果；實際應用於臺灣土雞種系遺傳態
品多形性分析，彙合個體間與種系間微衛星核酸的絕對長度多形性數據，準確
統計推論臺灣土雞種系親緣關係。
設計絕對長度核酸標準物MarkQoff 運用T7 促進子已知序列引子標定
IRDye 螢光，擴增已知序列dd[A/C/G]TP 終止產物的30~500 bp 混成物，具備
最少缺帶與最佳亂度。平板電泳6 檢體的單組引子微衛星核酸擴增產物分析，
依據配置市售標準物Licor SAGA 與絕對長度核酸標準物Aida MarkQoff；判
讀比較連續批次電泳的三重複長度數據統計結果顯示，MarkOff 部分均為相同
的正整數絕對長度數值，SAGA 部分則為不同的帶小數相對位移數值，且與實
際長度顯著偏差(標準誤差0.32)；再者，多組引子(ADL0176, ADL0181, ADL278,
LEI192)與單組引子的MarkOff 判讀絕對長度數值完全一致。製備絕對長度核
酸標準物MarkQoff 建立離心檻獲裝置Microcon YM30/50 純化收集30~500 bp
區段；標準物純液和0.5 倍稀釋液的電泳呈現相近訊號強度條帶，並明顯濾除
20~30 bp 區段。
技術平台應用於初步比較臺灣土雞種系遺傳分型12 品系48 雞隻的3 組高
度多形性共顯型微衛星核酸片段(ADL176、ADL278 及LEI192)，依據絕對長
度核酸標準物MarkQoff 判讀電泳影像條帶的絕對長度數據轉成二元字串，藉
運算軟體NTSYS 歸群火雞品系獨立分群(相似度0.06)，其餘品系互有摻雜。分
子變異數分析AMOVA 的品系內變異度7.864 大於品系間變異度2.132，符合
臺灣土雞五品系獨立歸群分析結果，遺傳變異顯示品系間均具顯著差異
(p<0.001)與群間品系顯著差異(p<0.001)，與烏骨雞呈現最大總變異度95.833。
關鍵字：微衛星核酸、遺傳態品、多形性、絕對長度核酸標準物。

ABSTRACT
The practical research tool of molecular marker techniques is applied
widely in genetic analysis with the frequent presence of high polymorphism and
reproducibility of microsatellite DNA markers among genomes of high class
organisms. With conventional size standards of sparse milestones, the data of
microsatellite genotyping, instead of non-decimal integer format of nature-size
data, is called as decimal numerical format of relative mobility ratio which in
turn results in data pooling difficulty and subsequent statistic error. The goal of
this thesis study is to design nature-size DNA standards (MarkQoff) in high
density milestones format for image data analysis software (Aida) towards an
integrated platform of genotyping task which calls non-decimal absolute-size
data of microsatellite DNA bands among multi collaborative parties for fully
compatible data pooling and analysis. Practically, the platform is initially
applied in genotyping the polymorphism among Taiwan native chickens on
local breeds for obtaining quality data in order to assist accurate statistical
analysis on the genetic relationships.
The design and preparation of MarkQoff nature-size DNA standards
exploits dd[A/C/G]TP DNA fragments of 30~500 bp mixture, with least missing
bands and optimal randomness, amplified with IRDye-T7 primer on known
sequence template for subsequent purification by HPLC and size cutoff
spin-filter. Comparative study over DNA standards of commercial Licor SAGA
and lab-made MarkQoff respectively revealed standard deviation of called
DNA sizes as of 0.32 versus 0.00 with six microsatellite samples which were
analyzed in triplicate runs of slab gel electrophoresis started at constant time
intervals of around 300 base-pair running distance. The preparation of
MarkQoff DNA demonstrated a 2-fold concentration of equal band signal
intensity with the purified 30~500 bp fraction by HPLC and size cutoff
spin-filter in which the 0~30 bp fraction has apparently disappeared.
In practice, the platform is applied on the preliminary comparison of genetic
relationships among 12 chicken breeds including control and Taiwan native
chickens with 48 genomic DNA samples concisely analyzed at 3 highly
polymorphic microsatellite loci of ADL176, ADL278, and LEI192. The
ix
translated absolute-size microsatellite DNA data of gel bands with MarkQoff
standards is transformed into binary 1/0 data string upon the presence and
absence of specified band. The NTSYS cluster analysis on 12 chicken breeds
indicated inter-breed crossing relationships except the independent cluster of
turkey with a similarity of 0.06. The AMOVA analysis demonstrated that the
intra-breed variance of 7.864 is greater than the inter-breed variance of 2.132
which is consistent with the results of independent cluster analysis with 5 local
breeds of Taiwan native chickens. The analysis results of genetic variation
revealed inter-breed significance (P < 0.001) and inter-cluster significance (P <
0.001) with various breeds combinations, whereas the Wugoo breed showed the
within-group sums of squares of 95.833.
Keywords: Microsatellite DNA, Genotyping, Polymorphism, Absolute-Size
DNA Standards.

目 錄
目 錄-----------------------------------------------------------------------------------------i
表目錄----------------------------------------------------------------------------------------iii
圖目錄----------------------------------------------------------------------------------------iv
關鍵簡首字---------------------------------------------------------------------------------vi
摘 要---------------------------------------------------------------------------------------vii
ABSTRACT------------------------------------------------------------------------------viii
第一章、微衛星核酸的遺傳分析與數據判讀--------------------------------01
第一節、遺傳分子標記類型--------------------------------------------------01
第二節、微衛星分子標記------------------------------------------------------03
第三節、微衛星分子標記的數據判讀議題-----------------------------04
第四節、絕對長度核酸標準物的技術平台任務目標---------------06
第五節、研究論文章節綱要--------------------------------------------------06
第二章、絕對長度核酸標準物設計----------------------------------------------09
第一節、前言-----------------------------------------------------------------------09
第二節、材料與方法-------------------------------------------------------------11
一、質體核酸製備-----------------------------------------------------11
二、平板電泳型絕對長度核酸標準物製備------------------12
三、毛細電泳型絕對長度核酸標準物製備------------------18
第三節、結果-----------------------------------------------------------------------20
一、平板電泳型絕對長度核酸標準物-------------------------20
二、毛細電泳型絕對長度核酸標準物-------------------------25
第四節、討論-----------------------------------------------------------------------27
一、絕對長度核酸標準物MarkQoff---------------------------27
二、絕對長度核酸標準物製備------------------------------------28
三、自動影像數據分析Aida 系統-------------------------------28
第三章、絕對長度核酸標準物製備------------------------------------------------31
第一節、前言-----------------------------------------------------------------------31
ii
第二節、材料與方法-------------------------------------------------------------32
一、高效能液相層析純化系統------------------------------------32
二、離心過濾檻物收集裝置----------------------------------------33
第三節、結果-----------------------------------------------------------------------34
一、高效能液相層析--------------------------------------------------34
二、離心過濾裝置------------------------------------------------------37
第四節、討論-----------------------------------------------------------------------39
一、高效能液相層析--------------------------------------------------40
二、離心過濾裝置------------------------------------------------------40
三、純化參數的改善--------------------------------------------------40
第四章、微衛星核酸絕對長度的遺傳態品應用-----------------------------43
第一節、前言-----------------------------------------------------------------------43
一、當地品種-------------------------------------------------------------43
二、臺灣土雞-------------------------------------------------------------44
三、微衛星分子標記與土雞遺傳分型--------------------------44
第二節、材料與方法--------------------------------------------------------------46
一、實驗動物族群------------------------------------------------------46
二、雞隻核酸來源------------------------------------------------------46
三、微衛星基因座------------------------------------------------------46
四、微衛星核酸片段聚合酶鏈鎖反應及電泳---------------48
五、分子遺傳資料庫分析系統------------------------------------50
第三節、結果------------------------------------------------------------------------50
一、微衛星核酸片段平板膠體電泳分析----------------------50
二、微衛星核酸片段絕對長度數據多形性分析------------52
第四節、討論------------------------------------------------------------------------60
一、臺灣土雞種系遺傳分型----------------------------------------60
二、微衛星核酸的計量性狀基因座分析應用---------------61
第五章、微衛星核酸絕對長度的整合數據與分析系統------------------63
第一節、絕對長度核酸標準物技術平台發展---------------------------63
第二節、微衛星核酸絕對長度的畜產種原遺傳分析應用--------64
第三節、微衛星核酸絕對長度的遺傳態品應用----------------------65
參考文獻------------------------------------------------------------67

朱幸男。2001。臺灣畜牧產業未來經營策略與發展方向。中畜會誌 30(4)：
67-79。
陳志峰、李淵百、范揚廣、黃三元、黃暉煌。1994。臺灣土雞種原保存。中
畜會誌 23(3)：339~346。
黃崇隆。2003。自動化分析電泳圖與基因微陣生物影像。碩士論文。台灣大
學資訊工程研究所。
蘇浚宏。2002。臺灣土雞種系之微衛星多形性遺傳差異分析。碩士論文。中
國文化大學生物科技研究所。
Botstein, D., White, R.L., Skolnick, M. and Davis, R.W. (1980) Construction
of a genetic linkage map using Restriction Fragment Length
Polymorphisms. American Journal of Human Genetics, 32:314-331.
Bruland, O., Almqvist, E. W., Goldberg, Y. P., Boman, H., Hayden, M. R., and
Knappskog, P. M. (1999) Accurate determination of the number of CAG
repeats in the Huntington disease gene using a sequence-specific internal
DNA standard. Clin Genet, 55:198-202.
Cheng H.H. (1997) Mapping the chicken genome. Poultry Science, 76:
1101-1107
Delmotte, F., Leterme, N., and Simon, J. C. (2001) Microsatellite allele sizing:
difference between automated capillary electrophoresis and manual
technique. BioTechniques, 31:810-818.
Diwan, N., and Cregan, P. B. (1997) Automated Sizing of Fluorescent-labeled
Simple Sequence Repeat (SSR) Markers to Assay Genetic Variation in
Soybean. Theor. Appl. Genet., 95:723-733.
Haberl, M. and Tautz, D. (1999) Comparative allele sizing can produce
inaccurate allele size differences for microsatellites. Molecular Ecology, 8:
1347-1350.
Harker, N. (2001) Collection, reporting and storage of micro- satellite
genotype data. Plant Genotyping: the DNA Finger- printing of Plants,
17:251-264.
Hillel, J., Groenen, M.A., Tixier-Boichard, M., Korol, A.B., David, L.,
Kirzhner, V.M., Burke, T., Barre-Dirie, A., Crooijmans, R.P., Elo, K.,
Feldman, M.W., Freidlin, P.J., Maki-Tanila, A., Oortwijn, M., Thomson,
P., Vignal, A., Wimmers, K., Weigend, S. (2003) Biodiversity of 52
chicken populations assessed by microsatellite typing of DNA pools.
Genet Sel Evol., 35(5):533-57.
Jouquand, S., Cheron, A., and Galibert, F. (1999) Microsatellite Analysis
Using a Two-Step Procedure for Fluorescence Labeling of PCR Products.
BioTechniques, 26:902-905.
Kaiser, M.G., Yonash, N., Cahaner, A., Lamont, S.J. (2000) Microsatellite
polymorphism between and Within broiler population. Poultry Science,
79: 626-628.
Kiba, Y. and Baba, Y. (1999) Unusual capillary electrophoretic behavior of
triplet repeat DNA. J. Biochem. Biophys. Methods, 41:143-151
Martin, G., Kenneth JF., Yeva B., Uwe DN., Kurt RY., Paul DR., Reb JRII.,
and John CG. (2002) Ion-pair reversed-phase high-performance liquid
參考文獻 68
chromatography analysis of oligonucleotides: Retention prediction.
Journal of Chromatography A, 958: 167-182.
Michael, L. M., Jing, L., and Richard, A. G. (1996) Electrophoretically
Uniform Fluorescent Dyes for Automated DNA Sequencing. Science,
271:1420-1422.
Mueller, U. G., Wolfenbarger, L. L. (1999) AFLP genotyping and
fingerprinting. Trends Ecol Evol., 14:389-394.
Philippe, J. and Lagoda, J.L. (1996) Microsatellite, from molecules to
populations and back. Trends in Ecology & Evolution, 11(10): 424-429.
Romano, M.N., Weigendt, S. (2001) Analysis of genetic relationships between
various populations of domestic and jungle fowl using microsatellite
markers. Poult Sci., 80(8):1057-63.
Rosenblum, B., Oaks, F., Menchen, S., and Johnson, B. (1997) Improved
single-strand DNA sizing accuracy in capillary electrophoresis. Nucleic
Acids Research, 25: 3925-3929.
Rosenberg, N.A., Burke, T., Elo, K., Feldman, M.W., Freidlin, P.J., Groenen,
M.A., Hillel, J., Maki-Tanila, A., Tixier-Boichard, M., Vignal, A.,
Wimmers, K., Weigend, S. (2001) Empirical evaluation of genetic
clustering methods using multilocus genotypes from 20 chicken breeds.
Genetics,159(2):699-713.
Schlotterer, C. (1998) Microsatellites. Molecular genetic analysis of
populations, 7: 237-261.
Schwengel, D. A., Jedlicka, A. E., Nanthakumar, E. J., Weber, J. L., and
Levitt, R. C. (1994) Comparison of fluorescence-based semi-automated
genotyping of multiple microsatellite loci with autoradiographic
techniques. Genomics, 22:46-54.
Sunnucks P. (2000) Efficient genetic markers for population biology. Trends
in Ecology & Evolution, 15(5): 199-203.
Tautz, D. (1989) Hypervariability of simple sequences as a general source for
polymorphic DNA markers. Nucleic Acids Research, 17:6463–6471.
Vanhala, T., Tuiskula-Haavisto, M., Elo, K., Vilkki, J., Maki-Tanila, A. (1998)
Evaluation of genetic variability and genetic distances between eight
chicken lines using microsatellite markers. Poult Sci., 77(6):783-90.
Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de Lee, T., Hornes, M.,
Frijtens, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. (1995)
AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research,
23:4407-4414
Welsh, J., and M. McClelland. (1990) Fingerprinting genomes using PCR
with arbitrary primers. Nucleic Acids Research, 18:7213–7218.
Williams, L. C., Hegde, M. R., Herrera, G., Stapleton, P. M., and Love, D. R.
(1999) Comparative semi-automated analysis of (CAG) repeats in the
Huntington disease gene: Use of internal standards. Molecular and
Cellular Probes, 13: 283 - 289.
Williams, J.G., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V.
(1990) DNA polymorphisms amplified by arbitrary primers are useful as
genetic markers. Nucleic Acids Research, 18:6231-6235.
Wimmers, K., Ponsuksili, S., Hardge, T., Valle-Zarate, A., Mathur, P.K.,
Horst P. (2000) Genetic distinctness of African, Asian and South
American local chickens. Animal Genetics, 31:159-164.