臺灣博碩士論文加值系統

鮪魚為國內重要的遠洋漁獲物，台灣常見魚種有黑鮪﹙Thunnus thynnus；bluefin tuna﹚、長鰭鮪﹙T. alalunga；albacore﹚、黃鰭鮪﹙T. albacares；yellowfin tuna﹚及大目鮪﹙T. obesus；bigeye tuna﹚等四種。鮪魚屬高經濟價值之魚類，魚種不同，價格差異很大；黑鮪的賣價最高，是鮪類經濟價值最高的魚種，其他平價之鮪魚生魚片大多為淺色肉之黃鰭鮪與大目鮪；而傳統鮪罐加工則以賣相較佳的白色肉長鰭鮪為主。為建立台灣常見遠洋鮪魚魚種新鮮原料之基因鑑定，本實驗將應用基因技術中PCR-RFLP ﹙polymerase chain reaction — restriction fragment length polymorphism﹚，配合直接定序法（sequencing），分析鮪魚魚種之基因圖譜；進而開發出利用基因探針快速辨別冷凍、新鮮及加工罐藏品之方法。
在海域族群的差異性方面，除T. thynnus之採樣侷限於北太平洋及北大西洋外，其餘三魚種之檢體均來自不同漁獲區，橫跨北半球三大洋，實驗得知T. obesus經PCR增幅之粒線體DNA中cytochrome b gene 358 bp片段與其他魚種相異度（divergence）相差最大，有最高4.235%的差異；而T. thynnus與T. alalunga相異度最小，相似度達98.687%至99.674%。經由序列分析四種鮪魚差異後，選得特異性切位限制酶包括Bsp1286 I、Hinc II及Rsa I，經Bsp1286 I作用後可將T. thynnus序列片段切割成283 bp與75 bp兩段，對其他魚種則無反應切位；Hinc II作用後除對T. obesus沒反應外，對其他三魚種皆有共同的作用位置，分解成198 bp、150 bp與10 bp三段；而Rsa I對T. albacares與T. obesus序列之切位相同，可切割成284 bp與74 bp兩段；對T. thynnus切成256 bp與102 bp兩段；而對T. alalunga則切割成256 bp、74 bp及28 bp三段，經由三組限制酶作用並交叉比對，在DNA電泳圖上可快速、確實的對四個鮪屬魚種作出區分。此外亦分析市售鮪魚生魚片種類，發現業者並無以低價鮪魚冒充高價黑鮪腹肉生魚片之情形。
加工品部分針對不同需求設計了7組引子對作PCR增幅反應，並注入不同調味液，分別以121℃/15 min與115℃/30 min模擬罐頭的幾種加工條件。然而DNA降解情形嚴重，只有CbBRs126L/H與CbHi146L/H兩組引子對順利增幅出PCR產物，分別為126 bp與146 bp的小片段目標序列，之後同樣以Bsp1286 I、Hinc II及Rsa I三組限制酶將模擬加工品作切位的交叉分析，可在電泳圖上成功判讀四魚種特殊的片段多型性。繼而檢驗台灣市售10件鮪罐產品，除2件產品無法順利增幅目標序列外，其他8件產品原料魚種分別驗出為6件黃鰭鮪與2件長鰭鮪，對國內肉類加工品之仿偽鑑定技術提供具體的學術參考依據。

Tuna is an important pelagic capture in Taiwan, the familiar species in Taiwan are bluefin tuna (Thunnus thynnus), albacore (T. alalunga), bigeye tuna (T. obesus) and yellowfin tuna (T. albacares). Tuna is a higher-price fish, and different species of tuna are quite different in cost. Bluefin tuna is the highest-price Thunnus species, and other raw fillets in the market are usually light meat, like bigeye tuna and yellowfin tuna. Traditionally, the raw material of canned tuna species is mainly white meat albacore. In order to establish the gene identification of fresh meat of familiar tuna species in Taiwan, in this study we used the directed sequencing and the polymerase chain reaction — restriction fragment length polymorphism (PCR-RFLP) technology to determine the genetic variation of four Thunnus species. Then we developed gene probes to identify the species of frozen, fresh and canned tuna meat.
In the part of the diversity of sea areas and groups, T. thynnus was obtained from northern Pacific Ocean and northern Atlantic Ocean, and other three species were from northern Pacific Ocean, northern Atlantic Ocean and Indian Ocean. Judging from the data of this study, the 358 bp fragment of the mitochondrial cytochrome b gene in T. obesus was mostly different from other species, there was the highest divergence (4.235%). And the highest similarity was between T. thynnus and T. alalunga (98.687%∼99.674%). After analyzing the difference of sequence in four Thunnus species, we chose three restriction enzymes with specific cutting sites, including Bsp1286 I, Hinc II and Rsa I. The restriction enzyme Bsp1286 I cleaved the 358 bp fragment to 283 bp and 75 bp in T. thynnus, and there was no any cutting site in other three species. Hinc II could cleave the 358 bp fragments of T. thynnus, T. alalunga and T. albacares to separate into 198 bp, 150 bp and 10 bp, but there was no cutting site in T. obesus. And then, Rsa I cleaved tuna fishes both T. thynnus and T. alalunga into fragments of 256 bp and 102 bp, it was different from the fragments of 284 bp and 74 bp in T. albacares and T. obesus. The polymorphic pattern in the DNA electrophoretic gel could identify four fresh Thunnus species precisely and quickly. Furthermore, we analyzed the 12 raw fillets of tuna sold in the market and found that there was no case to personate high-price T. thynnus by other tuna species.
In the part of canned product, we designed 7 pairs of primer for PCR amplifying. We added different sauces into tuna meat and then heated at 121℃ for 15 min and 115℃ for 30 min to simulate the process of canning. However, the DNA was degraded seriously, there were only two pairs of primer CbBRs126L/H and CbHi146L/H could successfully amplify the PCR products, short fragments of 126 bp and 146 bp, respectively. Then we used three restriction enzymes Bsp1286 I, Hinc II and Rsa I to analyze the cutting sites of procssed products and could differentiate the diversity between four species by the DNA electrophoretic map. Last, we analyzed 10 commercial canned products sold in the market, except 2 samples were failure in amplifying the target sequence, other 8 samples including 6 cases of T. albacares and 2 cases of T. alalunga were determined successfully. Therefore, this study could provide useful and academic technique, in identifying the species of processed tuna meats.

名詞縮寫對照表……….……………………………………………..Ⅰ
中文摘要………………...……………………………………………Ⅱ
英文摘要…………………..……………………………………….…Ⅳ
第一章、 文獻整理
一、 鮪屬（Thunnus）魚種之分類與漁場分布
（一） 鮪屬魚種之分類…..……………………………………...2
（二） 鮪屬魚種之漁場分布…..……………………………...…2
二、 我國遠洋鮪魚業及鮪罐加工之概況
（一） 我國遠洋鮪漁業之發展..………………………………...5
（二） 鮪罐加工之概況………..………………………………..5
三、 一般鑑定魚種之方法
（一） 以外觀特徵來判別魚種..………………………………..7
（二） 以蛋白質分析方法來鑑定魚種..………………………..8
四、 利用mtDNA鑑定物種
（一） mtDNA之介紹…………………..……………………...11
（二） 以mtDNA鑑定物種之優點……..……….…………….11
（三） mtDNA中cytochrome b gene之介紹…..……………..12
五、 應用分子生物技術─PCR技術鑑別魚種
（一） RAPD………………………………………..…………..14
（二） PCR-SSCP……………………………………..…….….15
六、 PCR-RFLP技術於基因型態之分析
（一） PCR-RFLP於物種分類上之應用……………..………..17
（二） PCR-RFLP於醫學上之應用…………………...……….18
（三） PCR-RFLP於加工食品上之應用………………..……..19
第二章、 台灣常見鮪屬魚種cytochrome b gene序列分析及PCR-RFLP鑑種技術之建立
一、 前言…………………………………………………………….23
二、 材料與方法
（一） 實驗材料………………………………………………………24
（二） 實驗方法
1. 粗萃DNA…………………………………………………….24
2. 選擇引子（primer）…………………………………………26
3. mtDNA中cytochrome b gene片段的大量增幅……………27
4. PCR產物cytochrome b gene片段之定序…………………..30
5. Cytochrome b gene片段資料分析…………………………..30
6. 限制酶分析PCR產物……………………………………….31
三、 結果
（一） 冷凍鮪魚之DNA粗萃………………………………………..33
（二） Cytochrome b gene序列片段之比較及分析…………………33
（三） 限制酶切位分析………………………………………………35
（四） 市售鮪魚生魚片之魚種檢測…………………………………36
四、 討論
（一） mtDNA中cytochrome b gene之序列特性……………….…..37
（二） 利用PCR-RFLP技術分析cytochrome b gene以鑑定魚種…38
圖表……………………………………………………………………40
第三章、 用PCR-RFLP技術分析鮪罐之加工條件及作為市售鮪罐魚種之鑑定
一、 前言……………………………………………………………..61
二、 材料與方法
（一） 實驗材料………………………………………………………62
（二） 實驗方法………………………………………………………62
三、 結果
（一） 加工條件對DNA粗萃之影響………………………………..65
（二） 選擇引子之適用性……………………………………………65
（三） PCR-RFLP鑑種技術之建立………………………………….66
（四） 市售鮪罐加工品之魚種檢測…………………………………67
四、 討論
（一） 加工條件影響DNA之降解情形……………………………..69
（二） 以PCR-RFLP檢測加工產品原料來源之可行性……………71
圖表……………………………………………………………………73
參考文獻……………………………………………………………...82
附錄……………………………………………………………………92

任曉晶。2002。養殖鰻及其加工品之魚種基因鑑定法探討。國立台灣海洋大學食品科學研究所碩士論文。基隆。
吳清熊、邱思魁。1996。水產食品學。國立編譯館。台北。
沈世傑。1993。鯖亞目，鯖科。台灣魚類誌，pp. 554-559。國立台灣大學動物學系。台北。
林榮輝。1998。進階版生物技術，pp. 246-250。臺大醫院出版。台北。
胡興華。2000。台灣漁業發展與作業漁場－鮪漁業篇。鮪魚年鑑，pp. 1-27。台灣區遠洋鮪漁船魚類輸出業同業公會。台北。
馬保之。1981。水產品罐頭。罐頭食品大全，pp. 696-732。食品工業發展研究所。新竹。
張鶴霖。1997。台北市生鮮鮪魚市場區隔化之研究。國立台灣海洋大學漁業經濟研究所碩士論文。基隆。
傅新輔。2000。鮪漁業常見之漁獲對象。鮪魚年鑑，pp. 215-226。台灣區遠洋鮪漁船魚類輸出業同業公會。台北。
黃鵬林、王愛玉、劉瑞芬、莊榮輝。2001。生物技術核心實驗。生物技術方法，卷一。國立台灣大學生物技術研究中心。台北。
楊永正。2000。巨分子序列分析研習會講義。國家衛生研究院。台北。
賴滋漢、金安兒、柯文慶。1992。食品加工學方法篇，pp. 306-309。精華出版社。台中。
賴滋漢、金安兒。1991。水產罐頭。食品加工學製品篇。精華出版社。台中。
謝喻文。2001。利用細胞色素b基因之PCR技術和基因序列作為紅鰭多紀魨和橫紋多紀魨原料及其加工品之魚種鑑定。國立台灣海洋大學食品科學研究所碩士論文。基隆。
蘇文慧。2001。巨分子序列分析基礎課程講義。國家衛生研究院。台北。
Alvarado, B. J. R., Naseri, I. and Ely, B. 1997. Orthodox and unorthodox phylogenetic relationships among tunas revealed by the nucleotide sequence analysis of the mitochondrial DNA control region. Journal of Fish Biology 50: 540-554.
Antointte, J. P. and Greg, S. S. 2001. Molecular phylogeny of the Chipmunks inferred from mitochondrial cytochrome b and cytochrome oxidase II gene sequences. Molecular Phylogenetics and Evolution 20 (3): 335—350.
AOAC. 1997. Official Methods of Analysis. In Association of Official Analysis Chemists, 16 Edition. Washington, DC.
Appleyard, S. A., Ward, R. D. and Grewe, P. M. 2002. Genetic stock structure of bigeye tuna in the Indian Ocean using mitochondrial DNA and microsatellites. Journal of Fish Biology 60: 767-770.
Bartlett, S. E. and Davidson, W. S. 1991. Identification of Thunnus tuna species by the polymerase chain reaction and direct sequence analysis of their mitochondrial cytochrome b gene. Canadian Journal Fisheries Aquatic Scence 48: 309-317.
Brown, W. G., Gadaleeta, G., Pepe, G., Saccone, C. and Sbisa, E. 1986. Structural conservation and variation in the D-loop containing region of vertebrate mitochondrial DNA. Journal of Molecular Biology 192: 503-511.
Calvo, J. H., Zaragoza, P. and Osta, R. 2001. A quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment. Journal of Animal Science 79: 2108-2112.
Carrera, E., Garcia, T., Cespedes, A., Gonzalez, I., Sanz, B., Hernandez, P. E. and Martin, R. 1998. Identification of Atlantic salmon (salmo salar) and rainbow trout (Oncorhynchus mykiss) by using polymerase chain reaction amplification and restriction analysis of the mitochondrial cytochrome b gene. Journal of Food Protection 61 (4): 482-486.
Carter, C. G., Seeto, G. S., Smart, A., Clarke, S. and Barneveld, R.J. 1998. Correlates of growth in farmed juvenile southern bluefin tuna Thunnus maccoyii (Castelnau). Aquaculture 161: 107-119.
Cespedes, A., Garcia, T., Carrera, E., Gonzalez, I., Fernandez, A., Hernandez, P. and Martin, R. 1999. Application of polymerase chain reaction－single strand conformational polymorphism (PCR-SSCP) to identification of flatfish species. Journal of AOAC International 82 (4): 903-907.
Cespedes, A., Garcia, T., Carrera, E., Gonzalez, I., Fernandez, A., Asensio, L., Hernandez, P. and Martin, R. 2000. Genetic differentiation between sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR-RFLP analysis of a 12S rRNA gene fragment. Journal of the Science of Food and Agriculture 80: 29-32.
Christensen, J. J., Peter, G. S. and Brita, B. 1995. Moraxella (Branhamella) catarrhalis: Restriction enzyme analysis typing with Hinf I, Hae III and Pst I. FEMS Immunology and Medical Microbiology 12: 43-46.
Collette, B. B. and Nauen, C. I. 1983. An annotated and illustrated cataloque of tunas, mackerels, bonitos and related species know to date. Scombrids of the world. In FAO Species Catalogue, Volume 2. Food and Agriculture Organization of USA.
Crockford, T. and Johnston, I. 1995. Isolation of unstable myosins and the analysis of light chains by capillary electrophoresis. Analysis of Biochemistry 231: 20-26.
Desalle, R. and Birstein, V. J. 1996. PCR identification of black caviar. Nature 361: 197-198.
Elvevoll, E. O., Sorensen, N. K., Osterud, B., Ofstad, R. and Martinez, I. 1996. Processing of marine foods. Meat Science 43 (8): 265-275.
Etienne, M., Fleurence, J., Rehbin, H., Kundiger, R., Yman, I. M., Frem, M., Craig, A., Mackie, I. M., Jessen, F., Smelt, A. and Luten, J. 1999. A standardized method of identification of raw and heat-processed fish by urea isoelectric focusing: A collaborative study. Electrophoresis 20: 1923-1933.
Garrett, R. H. and Grisham, C. M. 1999. Biochemistry, 2nd Edition. Saunders College Publishing. USA.
Gasser, R. B. and Chilton, N. B. 1995. Characterisation of taeniid cestode species by PCR-RFLP of ITS2 ribosomal DNA. Acta Tropica 59: 31-40.
Gray, W. M. 1989. Origin and evolution of mitochondrial DNA. Annual Review of Cell Biology 5: 25-50.
Hashimoto, K., Watabe, S., Nakagawa, T. and sorita, K. 1984. Electrophoretic identification of three subspecies of the genus Lagocephalus pufferfish. Bulletin of the Japanese Society of Scientific Fisheries 50: 115-118.
Haward, M. and Bergin, A. 2000. Taiwan’s distant water tuna fisheries. Marine Policy 24: 33-43.
Hsieh, H. M., Chiang, H. L., Tsai, L. C., Lai, S. Y., Huang, N. E., Linacre, A. and Lee, J. C. 2001. Cytochrome b gene for species identification of the conservation animals. Forensic Science International 122: 7-18.
Hsieh, Y. W., Shiu, Y. C., Cheng, C. A., Chen, S. K. and Hwang, D. F. 2002. Identification of toxin and fish species in cooked fish liver implicated in food poisoning. Journal of Food Science 67 (3): 948-952.
Hung, T. S., Marshall, M. R. and Wei, C. I. 1995. Identification of red snapper (Lutjanus campechanus) using electrophoretic techniques. Journal of Food Science 60: 279-283.
Hwang, D. F., Hsieh, Y. W., Shiu, Y. C., Chen, S. K. and Cheng, C. A. 2002. Identification of tetrodotoxin and fish species in a dried dressed fish fillet implicated in food poisoning. Journal of Food Protection 65 (2): 389-392.
Innis, M. A. and Gelfand, D. H. 1990. Optimization of PCR. In PCR Protocols－A Guide to Methods and Applications. pp.3-12. Academic Press Inc., New York.
Ishida, K., Zhu, B. -L., Sakoda, S., Quan, L., Oritani, S., Fujita, M. Q. and Maeda, H. 2000. Significance of DNA analysis for determination of ABO blood groups from hair and nail of decomposed human remain: a comparison with phenotyping by the absorption-elution method. Legal Medicine 2000 (2): 212-215.
Janusz, M., Bujnicki, M. R. and Leszek, R. 2001. Polyphyletic evolution of type II restriction enzymes revisited: two independent sources of second-hand folds revealed. Trends in Biochemical Sciences 26 (1): 9-11.
Jones, J. L. 1991. DNA probes: Application in the food industry. Trend Food Science Technology 2: 28-32.
Kocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V., Paabo, S., Villablanca, F. X. and Wilson, A. C. 1989. Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers. Proceedings of the National Academy of Science USA 86: 6196-6200.
Koolman, J. and Rohn, K. -H. 1996. Color Atlas of Biochemistry. pp. 237-241. Thieme Medical Publishers Inc., New York.
Kurihara, A., Tawata, M., Ikegishi, Y., Aida, K. and Onaya, T. 1999. The procedure of polymerase chain reaction — restriction fragment — single strand conformation polymorphism analysis by Hha I / Hinc II to detect mitochondrial DNA mutations. Life Sciences 64 (14): 1223-1230.
Lumini, E., Bosco, M. and Fernandez, M. P. 1996. PCR-RFLP and total DNA homology revealed three related genomic species among broad-host-range Frankia strains. FEMS Microbiology Ecology 21: 303-311.
Mackie, I. M. 1997a. Identify fish. In Food Authenticity. pp. 140-170. Blackie Academic and Professional. London.
Mackie, I. M. 1997b. Methods of identifying species of raw and processed fish. In Fish Processing Technology, 2nd Edition. pp. 160-199. Blackie Academic and Professional, London.
Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K., Yamada, J. and Shinmura, Y. 1999. A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science 51: 143-148.
Moritz, C., Dowling, T. E. and Brown, W. M. 1987. Evolution of animal mitochondrial DNA: Relevance for population biology and systematics. Annual Review of Ecology and Systematics 18: 262-269.
Nelson, D. L. and Cox, M. M. 1990. DNA repair. In Lehninger Principles of Biochemistry, Third Edition. pp. 949-958. Worth Publishers, New York.
Niwa, Y., Nakazawa, A., Margulies, D., Scholey, V. P., Wexler, J. B. and Chow, S. 2003. Genetic monitoring for spawning ecology of captive yellofin tuna (Thunnus albacares) using mitochondrial DNA variation. Aquaculture 218: 387-395.
Nozawa, H., Yamamoto, T., Uchihi, R., Yoshimoto, T., Tamaki, K., Hayashi, S., Ozawa, T. and Katsumata. 1999. Purification of nuclear DNA from single hair shafts for DNA analysis in forensic sciences. Legal Medicine 1999 (1): 61-67.
Partis, L., Croan, D., Guo, Z., Clark, R., Coldham, T. and Murty, J. 2000. Evaluation of a DNA fingerprinting method for determining the species origin of meats. Meat Science 54 (4): 369-376.
Ram, L. J., Ram, M. L. and Baidoun, F. F. 1996. Authentication of canned tuna and bonito by sequence and restriction site analysis of polymerase chain reaction products of mitochondrial DNA. Journal of Agriculture and Food Chemistry 44: 2460-2467.
Rehbein, H., Etienne, M., Jerome, M., Hattula, T., Knudsen, B., Jessen, F., Luten, J. B., Bouquet, W., Mackie, I. M., Ritchie, A. H., Martin, R. and Mendes, R. 1995. Influence of variation in methodology on reliability of the isoelectric focusing method of fish species identification. Food Chemistry 52: 193-197.
Rehbein, H., Kress, G. and and Schmidt, T. 1997. Application of PCR-SSCP to species identification of fishery products. Journal of the Science of Food and Agriculture 74: 35-41.
Ruby, J. D., Li, Y., Luo, Y. and Caufield, P. W. 2002. Genetic characterization of the oral Actinomyces. Archives of Oral Biology 47: 457-463.
Russell, V. J., Hold, G. L., Pryde, S. E., Rehbean, H., Quinteiro, J., Rey, -M. M., Sotelo, C. G., Santos, A. T. and Rosa, C. 2000. Use of restriction fragment length polymorphism to distinguish between salmon species. Journal of Agriculture and Food Chemistry 48: 2184-2188.
Saitoh, K. 1998. Genetic variation and local differentiation in the Pacific cod Gadus macrocephalus around Japan revealed by mt RNA and RAPD markers. Fisheries Science 64 (5): 673-679.
Sato, T., Matsuyama, J., Takahashi, N., Sato, M., Johnson, J., Schachtele, C. and Hoshino, E. 1998. Differentiation of oral Actinomyces species by 16S ribosomal DNA polymerase chain reaction — restriction fragment length polymorphism. Archives of Oral Biology 43: 247-252.
Sezaki, K., Kuboshima, Y., Mitani, I., Fukui, A. and Watabe, S. 2001. Identification of chub and spotted mackerels with mitochondrial cytochrome b gene and its application to respective pelagic eggs fixed with formalin. Nippon Suisan Gakkaishi 67 (2): 17-22.
Shaper, E. G., Robinson, M., Yee, D., Candlin, J. D., Mines, R. and Hunkapiller, T. 1996. Sensitivity and selectivity in protein similarity searches: A comparison of Smith-Waterman in hardware to BLAST and FastA. Genomics 38 (2): 179-191.
Sotelo, C. G., Pineiro, C., Gallardo, J. M. and Perez-Martin, R. I. 1993. Fish species identification in seafood products. Trends in Food Science and Technology 4: 395-401.
Southern, S., Peter, J. S. and Andrew, E. D. 1988. Molecular characterization of cloned mitochondrial genome. Journal of Molecular Evolution 28: 32-42.
Tagliavini, J., Harrison, I. J. and Gandolfi, G. 1995. Discrimination between Anguilla anguilla and A. rostrata by polymerase chain reaction－restriction fragment length polymorphism analysis. Journal of Fish Biology 47: 741-743.
Takeyama, H., Chow. S., Tsuzuki, H. and Matsunaga, T. 2001. Mitochondrial DNA sequence variation within and between tuna Thunnus species and its application to species identification. Journal of Fish Biology 58: 1646-1657.
Taniguchi, N., and Toshio, M. 1979. Identification of European, American, and Japanese eels by lactate dehydrogenase and malate dehydrogenase isozyme patterns. Bulletin of the Japanese Society of Scientific Fisheries 45 (1): 37-41.
Taniguchi, N., Takahashi, J. and Konishi, Y. 1972. Studies on a biochemical method for identification of the European and Japanese freshwater eel. Bulletin of the Japanese Society of Scientific Fisheries 38 (6): 627-631.
Tzeng, C. S., Hui, C. F., Shen, S. C. and Huang, P. C. 1992. The complete nucleotide sequence of the Crossostoma lacustre mitochondrial genome: Conservation and variations among vertebrates. Nucleic Acids Research 20 (18): 4853-4858.
Unseld, M., Beyermann, B., Brandt, P. and Hiesel, R. 1995. Identification of the species origin of highly processed meat products by mitochondrial DNA sequences. PCR Methods Application 4: 241-243.
Williams, J. F. 1989. Optimization strategies for the polymerase chain reaction. BioTechniques 7 (7): 762-767.
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: 531-535.
Wilson, A. C., Cann, R. L., Carr, S. M., George, M., Gyllensten, U. B., Higughi, R. G., Palumbi, S. R., Prager, E. M., Sage, R. D. and Stoneking, M. 1985. Mitochondrial DNA and two perspectives on evolutionary genetics. Biological Journal of the Linnean Society 26: 375-400.
Wolf, C., Rentsch, J. and Hubner, P. 1999. PCR-RFLP analysis of mitochondrial DNA: A reliable method for species identification. Journal of Agriculture and Food Chemistry 47 (4): 1350-1355.
Wu, Y. -Y., Delgado, R., Costello, R., Sunderland, T., Dukoff, R. and Csako, G. 2000. Quantitative assessment of apolipoprotein E genotypes by image analysis of PCR-RFLP fragments. Clinica Chimica Acta 293: 213-221.