臺灣博碩士論文加值系統

準確且可靠的動物種類鑑別方法在過去幾十年已穩定地增加，特別是由於最近的食品恐慌以及起因於大量進行非法買賣瀕危物種而造成生物多樣性的全面危機。最近的食品恐慌（例如：牛的海綿狀腦病變、禽流感、口蹄疫，等等），一些不當的食品製造者、宗教原因、食品過敏反應和基因改造生物(GMOs)已經明顯地加深關於食品組成的大眾意識。基於擴增和分析DNA方面而言，一種被稱為物種分子鑑別領域的新穎生物科技學可提供出有效的解決辦法。以DNA為基礎方法是使用肉品中物種所擁有的特殊DNA序列和檢測這物種特殊序列的可能性。尤其，粒腺體cytochrome b（Cyt b）基因序列已被證明是對此目的有幫助的。許多研究顯示Cyt b用於族群分析可足夠去區別家禽和家畜之間的不同種類。就分子生物學和技術而言，隨著這快速改進「傳統」篩選DNA序列的發展，進而對於物種鑑定提出有趣地遠景研究。因此，微陣列技術現下被認為能對生物多樣性的監控打開新遠景。
在目前的研究中，我們以最少量的primer數目同時在全血或是骨骼肌的動物來源中去擴增出足夠數量且具有物種特異性的粒腺體DNA（mtDNA）片段。這些primers是藉由生物資訊軟體的幫助下，依照Cyt b基因中具高度同源性區域的序列分析結果去進行設計，並且對應於全部物種的每條primer最多只包含3個mismatchs。此universal primer sets長度為19 - 23 mer，此是在包括全部物種Cyt b的序列分析結果中以人工的方式進行設計。由此方法去設計出3組共5對的PCR泛用引子對（PCR universal primer sets）（定名為PAL primer sets），可用於擴增粒腺體Cyt b中的DNA片段產生出一短鏈的PCR片段（short PCR fragment SPF）長度從103 bp到116 bp。此PAL primer sets確知可以非常敏感的偵測到57種動物物種，包括家畜，家禽及其相關的種類。為了藉由雜交反應（hybridization）去鑑別出特殊的動物種類，於是從SPF序列的比對結果中，在PAL primer sets之間序列去得到大小為50 mer的序列，再由其中去獲得57個具特異性的探針（probes）。這些50 mer的探針在其5端的部分以aminolinker去進行修飾及合成，並且固定到特殊玻璃材質的晶片上而可獲得mtDNA晶片。每一個晶片的矩陣上包含57個物種的特異性寡核苷酸探針（specific-species oligonucleotide probes），2個控制組（control）的探針，以及1個空白組（blank）探針。此具物種特異性的Cyt b片段是藉由以螢光Cy3或Cy5修飾過的PAL universal primer sets去擴增出來。經由螢光標記過的目標物（target）與mtDNA晶片以雜交反應去產生出特殊的螢光訊號而可鑑別出動物的種類。因此物種種類的鑑定和確定物種數量是將不同動物種類的混合物經由複合式聚合酶鏈反應（multiplex PCR）來進行擴增，以及mtDNA晶片的螢光雜交反應去做確認。
在這項研究中，所使用的參考動物種類是由一般商業來源中獲得共13種不同種類的脊椎動物（C. moschata、G. gallu、S. camelu、A. cygnoides、M. gallopavo、C. familiaris、F. catus、M. musculus、O. cuniculus、B. taurus、R. norvegicus、S. scrof和O. aries）。其SPF是使用PAL universal primer set A-f、B-f和AB-r去擴增出C. moschata、G. gallus、S. camelus、A. cygnoides和M. gallopavo五種物種。F. catus, C. familiaris, M. musculus和O. cuniculus四種物種則是以PAL universal primer set C-f和C-r進行擴增。R. norvegicus, B. taurus, S. scrofa和O. aries是以PAL universal primer set D-f、E-f和DE-r進行擴增。在此研究中，13個動物的物種確認主要是使用PAL primer sets於PCR或是multiplex PCR來擴增粒腺體Cyt b基因的目標區段，接著透過mtDNA晶片的雜交反應去分析擴增出來的DNA片段。在結果方面，可清楚得知藉由multiplex PCR技術去偵測到這些動物中具物種特異性的粒腺體Cyt b基因片段，是能夠從混和物中去偵測到其中的單一物種或是同時去偵測2 - 5種物種。這些species-specific primer sets是可使用於PCR和multiplex PCR中，並且能夠有效和明確地擴增出全部種類的Cyt b基因。在每個目標基因中也可被觀察到具有強烈雜交反應的訊號。這些結果指出包含57個species-specific探針的50 mer寡核苷酸陣列能夠明確符合於他們相對應的目標基因。而在晶片上雜交點的訊號強度極限是去測試DNA模版（DNA template）以1︰1、1︰0.1和1︰0.01的比例混和來觀察雜交信號的改變。在此結果中1% B. taurus與1% S. camelus的雜交點所得到的訊號強度是足夠高過背景的訊號值。這個「物種鑑別」晶片的設計雖然僅是透過人工來選擇探針的方式，其所得到探針大體上是準確且可信的。如此的設計方式或許是最容易的方法去得到57個物種甚至是更多種類的的低密度晶片（low-density chip）。
在這些研究內顯示出50 mer mtDNA晶片對於家禽和家畜的物種是具有物種鑑別的能力。在mtDNA晶片內的粒腺體Cyt b序列是有能力做為哺乳動物的物種鑑別所使用的標誌（marker）。另外它是可以在目前所擁有的陣列組合中加入更多的基因，使得將來所發展出包含各種哺乳動物的特殊晶片其鑑別能力能有所提升。此方式所得到的陣列式鑑別晶片是直接而且簡單的。它特別是在生態學和農業方面呈現出一種新應用方法的重要想法。一旦晶片已可被設計完成並且是可使用的，任何一般的實驗室應該能在短時間內進行鑑別。對於更廣泛的用途和經濟方面兩者的相關而言，或許將可發展為「Biodiversity」晶片，其中可包含大量鑑別特徵（diagnostic features）去分辨出在細菌，地衣，軟體動物，昆蟲，黴菌，哺乳動物等等物種的重要種類。

The need for accurate and reliable methods for animal species identification has steadily increased during past decades, particularly with the recent food safety issues and the overall crisis of biodiversity primarily resulting from the huge ongoing illegal traffic of endangered species. Recent food scares (e.g. bovine spongiform encephalopathy, avian influenza, foot-and-mouth disease, etc.), malpractices of some food producers, religious reasons, food allergies and genetically modified organisms (GMOs) have tremendously reinforced public awareness regarding the composition of food products. A relatively new biotechnological field, known as species molecular identification, based on the amplification and analysis of DNA, offers promising solutions. This DNA-base method used is based on the presence of species-specific sequences of DNA in meat and the possibility of detecting such sequences specifically. In particular, the mitochondrial cytochrome b (Cyt b) gene sequence has proven helpful for this purpose. Numerous studies showed that Cyt b is adequate for the discrimination of different species of poultry and livestock with cluster analysis. With the fast improvements in molecular biology and technology, alternatives to ‘classical’ DNA sequencing are being developed that present interesting perspectives for species identification studies. Therefore, microarray technology can now open up new perspectives for biodiversity monitoring.
In the present study, we showed using primer number as possibly minimal to simultaneously amplify and quantify specific mitochondrial DNA (mtDNA) fragments of animal source in whole blood or skeleton muscle. These primers were designed according the alignment results of identifying highly homologous regions within the Cyt b gene by bioinformation software and each primer contained a maximum of three mismatches to all animal species wherever possible. The universal primer sets 19 - 23 mer in length was designed manually on an alignment including all Cyt b sequences. In this approach, 5 universal PCR primer pairs in 3 groups of universal primer sets (named PAL primer sets) generating a short PCR fragment (SPF) were designed for amplification of the DNA fragment of mitochondrial Cyt b ranging from 103 bp to 116 bp. The PAL primer sets permit ultrasensitive detection in 57 animal species, including livestock, poultry and relevant species. To identify specific animals by hybridization, 57 specific probes in size of 50 mer inter-primer sequences were deduced from the alignment of SPF sequences. These 50 mer probes modified with aminolinker at the 5’-end were synthesized and immobilized onto a chip slide glass to create the mtDNA Chip. Each matrix contained 57 specific-species oligonucleotide probes, two control probes, and one blank in one chip was duplicated. The specific-species Cyt b fragments were amplified by universal primer sets modified with fluorescent Cy3 or Cy5 at 5’-end. The fluorescent labeled-targets used to hybridize the mtDNA Chip to generate designed profiling for species identification. Species identification and quantification were tested from the mixtures of different animal species by the multiplex PCR and fluorescent hybridization of mtDNA Chip.
In this study, reference animal species were obtained from 13 different vertebrate animals (C. moschata, G. gallu, S. camelu, A. cygnoides, M. gallopavo, C. familiaris, F. catus, M. musculus, O. cuniculus, B. taurus, R. norvegicus, S. scrof, and O. aries) from commercial sources. The SPF were amplified using the PAL universal primer set A-f, B-f, and AB-r for C. moschata, G. gallus, S. camelus, A. cygnoides and M. gallopavo. F. catus, C. familiaris, M. musculus and O. cuniculus used the PAL universal primer set C-f and C-r. R. norvegicus, B. taurus, S. scrofa and O. aries used the PAL universal primer set D-f, E-f, and DE-r. The discrimination of 13 animal species was based on PCR or multiplex PCR amplification of target regions of mtDNA Cyt b genes using PAL primer sets, followed by analysis of the amplified DNA by hybridization with the mtDNA Chip. In the results, it was clear that the multiplex PCR technique can detect specific mitochondrial Cyt b gene fragment of these animal species from the mixture of either species alone or from mixed 2 - 5 animal species. These results indicate that the 50 mer oligonucleotide array containing 57 species-specific probes appears to be specific to their corresponding target genes. The limit of hybridized spots intensity on the chip had tested hybridization signals changed were observed with template DNA proportions using 1:1, 1:0.1, and 1:0.01. In this result, the hybridization signal intensity of the spots was significantly higher than the background signal when the B. taurus in 1% and S. camelus in 1%. The design of this ‘species identification’ chip was done by visually choosing probes, which were generally accurate and robust. This might be the easiest approach for a low-density chip including up to 57 species or more species.
In these studies showed that 50 mer mtDNA Chip had the discrimination power as for species identification in poultry and livestock. The mitochondrial Cyt b sequences in the mtDNA Chip would be promising markers for the species identification of mammals. It can be used more genes in combination to enhance redundancies and thus robustness of a specific chip including mammals in the future. This approach for a diagnostic microarray-chip is straightforward and simple. It represents an important idea for the application of a new method particularly in the field of ecology and agriculture. Once a chip has been designed and is available, any small laboratory should be able to carry out the diagnostics in a short time. Of broader use, and therefore economically more relevant, could be a ‘Biodiversity-Chip’, containing a large number diagnostic features to distinguish key species in the taxa of bacteria, lichen, molluscs, insects, fungi, mammals, etc.

Content
Chinese Abstract............................................................................................................. i
English Abstract.............................................................................................................. iv
Content............................................................................................................................. viii
Content of Figures........................................................................................................... xi
Content of Tables............................................................................................................. xiii
I. Research Background and Significance.................................................................... 1
1-1. The significance of mitochondrial DNA................................................................ 1
1-2. Mitochondrial DNA structure................................................................................. 1
1-3. Nuclear-mitochondrial interactions........................................................................ 2
1-4. The mitochondrial cytochrome b gene of mammals.............................................. 3
1-5. Outlook for the animal species identification......................................................... 5
1-6. Methods of protein analysis used in the animal species identification................... 8
1-7. Methods of DNA analysis used in the animal species identification..................... 10
1-7-1. Polymerase Chain Reaction........................................................................... 10
1-7-2. Restriction Fragment Length Polymorphisms............................................... 12
1-7-3. Single strand conformational polymorphism analysis.................................. 13
1-7-4. Random amplified polymorphic DNA (RAPD) analysis.............................. 14
1-7-5. Identification species using DNA microarrays.............................................. 14
1-7-6. Quartz crystal microbalance (QCM)............................................................. 16
II. Materials and Methods.............................................................................................. 25
2-1. Reagents.................................................................................................................. 25
2-2. Specimens of vertebrate animal.............................................................................. 25
2-3. Mitochondrial DNA extraction............................................................................... 25
2-4. Design of PCR primers........................................................................................... 26
2-5. Amplification of the fragment of the mitochondrial DNA Cyt b gene................... 27
2-6. Gel/PCR DNA extraction and purification............................................................. 27
2-7. Digestion of restriction enzymes............................................................................ 28
2-8. Cloning the DNA fragments of Cyt b gene............................................................ 28
2-9. Design of specific oligonucleotide microarray probes for species identification... 30
2-10. mtDNA Chip design and fabrication.................................................................... 31
2-11. Printing of the animal species-specific oligonucleotide probes on glass slides.... 31
2-12. mtDNA Chip Pre-Hybridization........................................................................... 32
2-13. Synthesis of Cy5-labeled or Cy3-labeled targets................................................. 32
2-14. Hybridization of the mtDNA Chip....................................................................... 33
2-14-1. Hybridization condition............................................................................... 33
2-14-2. Post-Hybridization washing........................................................................ 33
2-14-3. mtDNA Chip scanning and data analyses................................................... 34
2-15. Design of PCR primers for QCM......................................................................... 35
2-16. Amplification of the fragment of the mitochondrial DNA Cyt b gene for QCM.................................................................................................................... 35
2-17. Design of species-specific probes for QCM analysis........................................... 36
2-18. QCM measurement for species identification...................................................... 37
2-18-1. QCM apparatus............................................................................................ 37
2-18-2. Immobilization of the oligonucleotide probe on gold................................. 37
2-18-3. Hybridisation with purified PCR product................................................... 38
III. Result and Discussion............................................................................................... 58
3-1. The total DNA Extraction....................................................................................... 58
3-2. PCR amplification of target mtDNA Cyt b genes................................................... 58
3-3. Identification of animal species by RFLP............................................................... 60
3-4. Identification of animal species by mtDNA Chip................................................... 62
3-5. Detection sensitivity of 50 mer mtDNA array hybridization.................................. 63
3-6. PCR amplification of target mtDNA Cyt b genes using the BSO universal primer set................................................................................................................ 66
3-7. Direct monitoring of the single species-specific probe immobilization and hybridization of single species target...................................................................... 66
Conclusions...................................................................................................................... 80
References........................................................................................................................ 82

Content of Figures
Figure 1-1 This diagram summarizes the major features of mtDNA referred to in the text........................................................................................................ 18
Figure 1-2. The Principle of Quartz crystal microbalance (QCM)............................... 19
Figure 2-1 PAL universal primer sets and species-specific probes designed for animal species identification...................................................................... 39
Figure 2-2. Alignment pattern of Cyt b gene for the specific probes and universal primer pair design....................................................................................... 40
Figure 2-3 The 103 bp Cyt b PCR fragments of B. taurus sequencing electropherogram of pCR 2.1-TOPO vector............................................... 41
Figure 2-4 Probe arrangement of the mtDNA Chip for species identification............ 42
Figure 2-5 A schematic describing the oligonucleotide array detection assay............. 43
Figure 2-6 Fabrication of the mtDNA Chip................................................................. 44
Figure 2-7 Diagram of the current feature (A) defining the local background component.................................................................................................. 45
Figure 3-1 Agarose electrophoresis of mtDNA extractions extracted from all animal species skeleton muscle or whole blood in this study.................... 68
Figure 3-2 Agarose electrophoresis of PCR products amplified from the mitochondrial Cyt b gene of various species............................................. 69
Figure 3-3 Electrophoretic analysis of the mutiplex PCR products of the Cyt b gene fragment by using two restriction enzymes RsaI and StyI digestion.......... 70
Figure 3-4 Electrophoretic analysis of the multiplex PCR products of the Cyt b gene fragment in varying proportions by using two restriction enzymes RsaI and StyI digested on 3% agarose gel.................................................. 71
Figure 3-5 Electrophoretic analysis of the multiplex PCR products of the Cyt b gene fragment in mixed 4 - 5 animal species by using two restriction enzymes RsaI and StyI digested on 3% agarose gel................................... 72
Figure 3-6 Electrophoretic analysis of the PCR products of the Cyt b gene fragment by using two restriction enzymes RsaI and StyI digestion......................... 73
Figure 3-7 mtDNA Chip hybridization pattern of single species PCR product amplified by PAL universal primer for species identification.................... 74

Figure 3-8 mtDNA Chip hybridization pattern of the multiplex PCR products of B. taurus and S. camelus in varying proportions amplified by different universal primer set.................................................................................... 75
Figure 3-9 mtDNA Chip hybridization pattern of the multiplex PCR products of B. taurus and S. scrofa in varying proportions amplified by the same universal primer set.................................................................................... 76
Figure 3-10 mtDNA Chip hybridization pattern of the multiplex PCR products of mixed 4 - 5 animal species in the same proportions amplified by the PAL universal primer set............................................................................ 77
Figure 3-11 Agarose electrophoresis of PCR products amplified from the mitochondrial Cyt b gene of B. taurus, O. aries, and S. scrofa.................. 78
Figure 3-12 Frequency variations during the immobilization of the thiolated single species-specific probe directly onto the gold electrode of the quartz crystal and hybridization of single species target....................................... 79


Content of Tables
Table 1-1 Fate of light and heavy chain transcripts from mitochondrial DNA.......... 20
Table 1-2 Examples of forensic or food substrates for molecular analysis................ 21
Table 1-3 Various methodologies based on protein analysis for the identification of seafood........................................................................................................ 22
Table 1-4 Common molecular techniques useful for evolutionary studies, and selected examples in the Acari................................................................... 23
Table 2-1 List of 13 species in all 57 animal species for species identification used in this study................................................................................................ 46
Table 2-2 List of all 57 animal species for species identification and the PAL universal primer sets used for amplification of all 57 animal species in this study..................................................................................................... 47
Table 2-3 The PAL (poultry and livestock) universal primer sets used in this study............................................................................................................ 50
Table 2-4 Restriction fragment length polymorphism (RFLP) patterns (in bp for each band) of the PCR fragments from animal species generated after digestion with endonuclease enzymes........................................................ 51
Table 2-5 Oligonucleotide probes for identificaiton of all 57 animal species in the study........................................................................................................... 52
Table 2-6 The BSO (B. taurus, S. scrof, and O. aries) universal primer sets used for QCM..................................................................................................... 56
Table 2-7 Oligonucleotide probes for identificaiton of B. taurus, S. scrof, and O. aries in QCM analysis................................................................................ 57

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