臺灣博碩士論文加值系統

本研究旨在探討豬隻類胰島素生長因子乙型（insulin-like growth factor 2, IGF2）基因序列點突變對生產性能之影響。應用PCR擴增IGF2之DNA片段，經膠體純化（gel extraction）、連接（ligation）、轉型（transformation）與藍白菌落篩選（blue/white color screening）後，進行基因定序。比對NCBI （AY044828）公布序列，發現位於該基因第七內含子第162個鹼基上有一點突變，G→C （IGF27-G162C）。運用MS-PCR（Mutagenically separated PCR）原理設計合適引子與進行基因型鑑定，計有CC、CG與GG型。基因檢測檢定站完檢232頭藍瑞斯、68頭約克夏與434頭杜洛克豬種，GG基因型頻率分別0.86、1.47與12.21%。杜洛克公豬CC基因型較GG基因型者早達110 kg重（P=0.06）。不分品種比較，CC基因型豬隻有較佳平均隻日增重、較薄背脂與較高選拔指數之趨勢。再者，比較CC及GG純合型之雜交肉豬屠體性狀發現，CC基因型雜交閹公豬之小里肌肉率與較大第10-11肋腰眼肉面積之趨勢；而CC基因型雜交女豬，則有顯著較低脂肪率，以及較高後腿率、瘦肉率和主要部位肉率。結果顯示：豬IGF2第七內含子之第162個鹼基點突變， C→G，不利於生長與屠體性能表現， C交替基因是產肉性能有利基因。杜洛克、藍瑞斯與約克夏豬種之IGF2基因第三內含子第3072個鹼基點突變（IGF23-G3072A）之AA基因型頻率分別為97.47% （423/434）、77.59%（180/232）與72.06%（49/68）；其中藍瑞斯與約克夏僅各有一頭GG基因型，而杜洛克則無GG型。品種內不同IGF23-G3072A基因型豬隻生長檢定性能比較顯示，具AA基因型之杜洛克與藍瑞斯，以及AG基因型約克夏豬隻具較薄背脂、達110 kg重日齡較早，且有高的選拔指數之趨勢。同時，AA基因型雜交閹公豬有顯著較高之前腱肉率、里肌肉率和後腿肉率；AA基因型雜交女豬，則具較薄最後肋背脂厚度與較高後腿肉率。綜合上述，台灣豬種IGF23-3072A與IGF27-162C交替基因選留有利於豬生產性狀改進。

The purpose of this study was to evaluate the effect of point mutation in IGF2 gene on meat production related traits in pigs. The PCR product obtained from gDNA fragment amplification was then electrophoreses, followed by gel extraction, ligation, transformation, blue/white color screening and finally the sequencing procedure. After alignment with sequence data (AY044828) of porcine IGF2 gene published in NCBI, the obtained sequence revealed one point mutation (G→C) in position 162 of intron 7 (IGF27-G162C). The IGF27-G162C polymorphism was genotyped by mutagenically separated PCR (MS-PCR) method using appropriate designed primers and CC, CG and GG genotypes were observed. There were 232, 68 and 434 off-test Landrace, Yorkshire and Duroc pigs genotyped and the corresponding frequencies for GG genotype were 0.86、1.47 and 12.21%, respectively.
Within breed comparison, Duroc boars with CC genotype tended to reach 110 kg weight earlier (P=0.06). However, when pooled data was used, CC genotype boars also showed higher average daily gain, thinner backfat thickness and better selection index than those of boars with GG genotype. When comparison was made for carcass traits among crossbred hogs, castrates with CC genotype tended to show heavier tender loin (%) and loin eye area between the 10th and 11th ribs. Crossbred females with CC genotype had significant lower fat%, higher leg muscle%, higher lean mass and major lean mass, in terms of carcass weight. Results indicated that the IGF27-G162 allele had adverse effects on both growth and carcass performances, and the IGF27-162C allele was the favorable allele for the corresponding production performances in pigs. Within this study, the dominant genotype of IGF23-G3072A was AA, which accounted for 97.47% (423/434), 77.59% (180/232) and 72.06% (49/68) in Duroc, Landrace and Yorkshire off-test pigs, respectively. There was only one pig each with GG genotype in Landrace and Yorkshire breeds observed in this study. No Duroc pig with GG genotype was found. When comparison was conducted within breed, Duroc and Landrace pigs with AA genotype, and Yorkshire pigs with AG genotype showed thinner backfat thickness, reached 110 kg off-test weight earlier and higher selection index than those of other genotypes. Castrates with AA genotype tended to have higher shoulder shank muscle (%), loin muscle (%) and leg muscle (%). Crossbred females with AA genotype had significant thinner backfat thickness at the last rib and higher leg muscle (%) In conclusion, both IGF27-162C and IGF23-3072A would be the favorable alleles for production traits improvement in Taiwan pig industry.

目錄
摘要 I
Abstract III
誌謝 V
目錄 VI
圖表目錄 IX
壹、前言 1
貳、文獻回顧 3
一、豬隻第二號染色體上數量性狀基因座（QTL）對屠體性能之影響 3
二、豬隻類胰島素生長因子乙型（IGF2）基因介紹 5
（一）基因序列 5
（二）基因調控 10
（三）類胰島素生長因子系統 12
（四）豬隻IGF2表現量 15
（五）點突變 17
1.第二內含子（intron 2） 17
2.第三內含子（intron 3） 20
3.第八內含子（intron 8） 24
參、類胰島素生長因子乙型基因第七內含子（IGF2-intron7）點突變基因型分析試驗 25
一、材料與方法 25
（一）試驗動物 25
（二）基因組DNA萃取 25
（三）DNA定量 25
（四）基因點突變檢測方法 26
1.聚合酶鏈鎖反應-限制酵素片段多態性（PCR-RFLP） 26
(1)引子序列 26
(2)PCR反應液之配置 26
(3)PCR反應條件 26
(4)限制酵素切割作用 26
(5)膠體電泳 27
2.突變點拆離式聚合酶鏈鎖反應（MS-PCR） 27
(1)原理 27
(2)引子設計原則 27
（五）PCR產物純化 29
（六）PCR產物選殖 29
1.DNA黏接作用（ligation） 29
2.轉形作用（transformation） 31
3.質體DNA萃取 31
4.重組質體DNA確認 31
（七）PCR產物之核酸定序分析與序列比對 32
（八）統計分析 32
二、結果與討論 33
（一）IGF2-intron7點突變定序結果 33
（二）應用PCR-RFLP模式分析IGF2-intron7基因型 35
（三）應用MS-PCR模式分析IGF2-intron7基因型 36
1.引子與反應條件 36
(1)引子序列 36
(2)PCR反應液之配置 36
(3)PCR反應條件 36
2.MS-PCR引子專一性測定 37
3.PCR-RFLP與MS-PCR結果比對 39
（四）豬IGF2-intron7基因型對生產性狀之影響 40
肆、類胰島素生長因子乙型基因第三內含子（IGF2-intron3）點突變基因型分析試驗 50
一、材料與方法 50
（一）試驗動物 50
（二）試驗步驟 50
（三）統計分析 50
二、結果與討論 51
（一）應用MS-PCR模式分析IGF2-intron3基因型 51
1.引子與反應條件 51
(1)引子序列 51
(2)PCR反應液之配置 51
(3)PCR反應條件 51
2.MS-PCR引子專一性測定 52
（二）第三內含子點突變多態性定序結果 53
（三）豬IGF2-intron3基因型對生產性狀之影響 55
伍、豬IGF23-G3072A與IGF27-G162C基因型組合效應分析 65
一、對中央檢定公豬生長性能之影響 65
二、對雜交肉豬屠體性能之影響 68
陸、結論 72
參考文獻 76
附錄 86
作者簡介 87

李兆峯、林皓雯、戴珮珊、黃純純。2005。台灣養豬統計手冊，第2頁。行政院農業委員會，台北市。
劉桂蘭、蔣思文、熊遠著、鄭嶗、屈彥純。2003。IGF2基因PCR-RFLP多態性與脂肪沉積相關性狀的關聯分析。動物遺傳學 30：1107-1112。
Amarger, V., M. Nguyen, A. V. Laere, M. Braunschweig, C. Nezer, M. Georges, and L. Andersson. 2002. Comparative sequence analysis of the INS-IGF2-H19 gene cluster in pig. Mamm. Genome 13:388-398.
Andersson-Eklund, L., L. Marklund, K. Lundstrom, C. S. Haley, K. Andersson, I. Hansson, M. Moller, and L. Andersson. 1998. Mapping quantitative trait loci for carcass and meat quality traits in a wild boar × large white intercross. J. Anim. Sci. 76:694-700.
Andersson, L., and M. Georges. 2004. Domestic-animal genomics: deciphering the genetics of complex traits. Nature Reviews 5:202-212.
Baker, J., L. Jeh-Ping, E. J. Robertson, and A. Efstratiadis. 1993. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73-82.
Bayes-Genis, A., C. A. Conover, and R. S. Schwartz. 2000. The insulin-like growth factor axis. Circ. Res. 86:125-130.
Bell, A. C., and G. Felsenfeld. 2000. Methylation of a CTCF-dependent boundary controls imprinted expression of the IGF2 gene. Nature 405:482-485.
Bowen, R. A. R., B. Y. L. Wong, and D. E. C. Cole. 2004. Population-based differences in frequency of the transcobalamin II Pro259Arg polymorphism. Clin. Biochem. 37:128-133.
Buonomo, F. C., and J. Klindt. 1993. Ontogeny of growth hormone (GH), insulin-like growth factors (IGF-I and IGF-II) and IGF binding protein-2 (IGFBP-2) in genetically lean and obese swine. Domest. Anim. Endocrin. 10:257-265.
Carrodeguas, J. A., C. Burgos, C. Moreno, A. C. Sánchez, S. Ventanas, L. Tarrafeta, J. A. Barcelona, and M. O. López-Buesa. 2005. Incidence in diverse pig populations of an IGF2 mutation with potential influence on meat quality and quantity: An assay based on real time PCR (RT-PCR). Meat Sci. 71:577-582.
Catchpole, I. R., and W. Engström. 1990. Nucleotide sequence of a porcine insulin-like growth factor II cDNA. Nucleic Acids Res. 8: 6430.
Chan, P. C. R., B. Y. L. Wong, H. Ozcelik, and D. E. C. Cole. 1999. Simple and rapid detection of BRCA1 and BRCA2 mutations by multiplex mutagenically separated PCR. Clin. Biochem. 45:1285-1287.
Chen, Y. J., Q. B. Tang, and S. Q. Zou. 2005. Inactivation of RASSF1A, the tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma. World J. Gastroenterol. 11:1333-1338.
Crump, R. E., R. Thompson, C. S. Haley, and J. Mercer. 1997. Individual animal model estimates of genetic correlations between performance test and reproduction traits of Landrace pigs performance tested in a commercial nucleus herd. Anim. Sci. 65:291-298.
Daughaday, W. H., and P. Rotwein. 1989. Insulin-like growth factor I and II. peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr. Rev. 10:68-91.


De Koning, D. J., A. P. Rattink, B. Harlizius, J. A. M. van Arendonk, E. W. Brascamp, and M. A. M. Groenen. 2000. Genome-wide scan for body composition in pigs reveals important role of imprinting. Genetics 97:7947-7950.
De Koning, D. J., A. P. Rattink, B. Harlizius, M. A. M. Groenen, E. W. Brascamp, and J. A. M. van Arendonk. 2001. Detection and characterization of quantitative trait loci for growth and reproduction traits in pigs. Livest. Prod. Sci. 72:185-198.
Donohoe, G. G., M. Laadsonen, K. Pulkki, T. Ronnemma, and V. Kairisto. 2000. Rapid single-tube screening of the C282Y hemochromatosis mutation by real-time multiplex allele-specific PCR without fluorescent probes. Clin. Chem. 46:1540-1547.
Dull, T. J., A. Gray, J. S. Hayflick, and A. Ullrich. 1984. Insulin-like growth factor II precursor gene organization in relation to insulin gene family. Nature 310:777-781.
Dwyer, C. M., J. M. Fletcher, and N. C. Stickland. 1993. Muscle cellularity and postnatal growth in the pig. J. Anim. Sci. 71:3339-3343.
Endler, G., P. A. Kyrle, S. Eichinger, M. Exner, and C. Mannhalter. 2001. Multiplexed mutagenically separated PCR: simultaneous single-tube detection of the factor V R506Q (G1691A), the prothrombin G20210A, and the methyl-enetetrahydrofolate reductase A233V (C677T) variants. Clin. Biochem. 47:333-335.
Ewton, D. Z., S. L. Falen, and J. R. Florini. 1987. The type II insulin-like growth factor (IGF) receptor has low affinity for IGF-I analogs: pleiotypic actions of IGFs on myoblasts are apparently mediated by the type I receptor. Endocrinology 120:115-123.
Florini, J. R., D. Z. Ewton, and S. A. Coolican. 1996. Growth hormome and the insulin-like growth factor system in myogenesis. Endocr. Rev. 17:481-517.
Frater, A. J., C. C. Chaput, S. Beddows, J. N. Weber, and M. O. McClure. 2001. Simple detection of point mutation associated with HIV-1 drug resistance. J. Virol. Methods 93:145-156.
Gerrard, D. E., C. S. Okamura, M. A. M. Ranalletta, and A. L. Grant. 1998. Developmental expression and location of IGF-Ⅰand IGF-Ⅱ mRNA and protein in skeletal muscle. J. Anim. Sci. 76:1004-1011.
Hausman, G. J., D. R. Campion, and F. C. Buonomo. 1991. Concentration of insulin-like growth facters (IGF-I and IGF-II) in tissue of developing lean and obese pig fetuses. Growth Develop. Aging 55:43-52.
Hedley, P. E., A. M. Dalin, and W. Engströmm. 1989. Developmental regulation of insulin like growth factor II gene expression in the pig. Cell Biol. Int. Rep. 13:857-861.
Hermesch, S., B. G. Luxford, and H. -U. Graser. 2000. Genetic parameters for lean meat yield, meat quality, reproduction and feed efficiency traits and genetic correlations with production, carcase and meat quality traits. Livest. Prod. Sci. 65:261-270.
Hill, A. E., and D. R. FitzPatrick. 1998. MS-PCR assay to detect 677C-T mutation in the 5,10-methylenetetrahydrofolate reductase gene. J. lnger. Metab. Dis. 21:694-695.
Hirooka, H., D. J. de Koning, B. Harlizius, J. A. M. van Arendonk, A. P. Rattink, M. A. M. Groenen, E. W. Brascamp, and H. Bovenhuis. 2001. A whole-genome scan for quantitative trait loci affecting teat number in pigs. J. Anim. Sci. 79:2320-2326.
Hunt, C. C. J., J. E. Burley, C. M. L. Chapman, and J. P. Beilby. 1999. A high-throughput MS-PCR method of MADGE gels for ANG II type-1 receptor A1166C polymorphism. Physiol. Genomics 8:71-73.
Ishihara, K., N. Hatano, H. Furuumi, R. Kato, and T. Iwaki. 2000. Comparative genomic sequencing identifies novel tissue-specific enhancers and sequence elements for methylation-sensitive factors implicated in IGF2/H19 imprinting. Genome Res. 10:664-674.
Jeon, J. T., O. Carlborg, A. Tornsten, E. Giuffra, V. Amarger, P. Chardom, L. Andersson-Elisabetta, K. Andersson, L. Hansson, K. Lundstrom, and L. Andersson. 1999. A paternally expressed QTL affecting skeletal and cardiac muscle mass in pigs maps to the IGF2 locus. Nat. Genet. 11:131-135.
Jungerius, B. J., A. S. van Laere, M. F. W. Tepas, B. A. van Oost, L. Andersson, and M. A. M. Groenen. 2004. The IGF2-intron3-G3072A substitution explains a major imprinted QTL effect on backfat thickness in a Meishan × European white pig intercross. Genet. Res. 84:95-101.
Klenova, E. M., R. H. Nicolas, H. F. Paterson, A. F. Carne, C. M. Heath, G. H. Goodwin, P. E. Neiman, and V. V. Lobanenkov. 1993. CTCF, a conserved nuclear factor required for optimal transcriptional activity of chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms. Mol. Cell Biol. 13:7612-7624.
Knoll, A., L. Putnova, J. Dvorak, and S. Cepica. 2000. A NciI PCR-RFLP within intron 2 of the porcine insulin-like growth factor 2 (IGF2) gene. Anim. Genet. 31:150-151.
Kolarikova, O., L. Putnova, T. Urban, J. Adamek, A. Knoll, and J. Dvorak. 2003. Associations of the IGF2 gene with growth and meat efficiency in Large White pigs. J. Appl. Genet. 4:509-513.
Lamberson, W. R., J. A. Sterle, and R. L. Matteri. 1996. Relationships of serum insulin-like growth factor II concentrations to growth, compositional, and reproductive traits of swine. J. Anim. Sci. 74:1753-1756.
Lee, C. Y., F. W. Bazer, T. D. Etherton, and F. A. Simmen. 1991. Ontogeny of insulin-like growth factors (IGF-I and IGF-II) and IGF-binding proteins in porcine serum during fetal and postnatal development. Endocrinology 128:2336-2343.
Lee, J. C., L. C. Tsai, C. H. Chen, and J. G. Chang. 1996. ABO genotyping bymutagenically separated polymerase chain reaction. Forensic Sci. Int. 82:227-232.
Leighton, P. A., R. S. Ingram, J. Eggenschwiler, A. Efstratiadis, and S. M. Tilghman. 1995a. Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature 375:34-39.
Leighton, P. A., J. R. Saam, R. S. Ingram, C. L. Stewart, and S. M. Tilghman. 1995b. An enhancer deletion affects both H19 and IGF2 expression. Genes Dev. 9:2079-2089.
LeRoith, D., and C. T. Roberts. 2003. The insulin-like growth factor system and cancer. Cancer Letters 195:127-137.
Lewis, A., and A. Murrell. 2004. Genomic imprinting: CTCF protects the boundaries. Curr. Biol. 14:284-286.
Liu, Q., E. C. Thorland, J. A. Heit, and S. S. Sommer. 1997. Overlapping PCR for bidirectional PCR amplification of specific alleles: a rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes. Genome Res. 7:389-398.

Liu, Y., B. Sullivan, S. Chen, N. Buys, and P. K. Mathur. 2006. Effects of IGF2 gene on carcass and meat quality in Canadian swine. Page 13-18 in 8th World Congress on Genetics Applied to Livestock Production, Belo Horizonte, MG, Brazil.
Lockley, A. K., J. S. Bruce, S. J. Franklin, and R. G. Bardsley. 1996. Use of mutagenically separated PCR for the detection of the mutation associated with porcine stress syndrome. Meat Sci. 43:93-97.
Lockley, A. K., J. S. Bruce, S. J. Franklin, and R. G. Bardsley. 1997. Simultaneous detection of a sex-specific sequence and the Ryr1 point mutation in porcine genomic DNA. Meat Sci. 45:485-490.
Loebel, D. A. F., and P. P. L. Tam. 2004. Mice without a father. Nature 428:809-811.
Morison, I. M., and A. E. Reeve. 1998. Insulin-like growth factor 2 and overgrowth: molecular biology and clinical implications. Mol. Med. Today 4:110-115.
Myint, L., K. Ariyoshi, H. Yan, A. J. Frater, W. Auwanit, P. Pathipvanith, K. Yamada, M. Matsuda, T. Chiba, K. Fujita, M. McClure, J. N. Weber, and W. Sugiura. 2002. Mutagenically separated PCR assay for rapid detedction of M41L and K70R zidovudine resistance mutations in CRF01_AE (Subtype E) human immunodeficiency virus type 1. Antimicrob. Agents Ch. 46:3861-3868.
Nezer, C., L. Moreau, B. Brouwers, W. Coppieters, J. Detilleux, R. Hanset, L. Karim, R. Hanset, L. Karim, A. Kvasz, P. Leroy, and M. Georges. 1999. An imprinted QTL with major effect on muscle mass and fat deposition maps to the IGF2 locus in pigs. Nat. Genet. 21:155-156.

O’Dell, S. D., and I. N. M. Day. 1998. Molecules in focus insulin-like growth factor II (IGF-II). Int. J. Biol. Cell. Biol. 30:767-711.
Ohlsson, R., R. Renkawitz, and V. Lobanenkov. 2001. CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends Genet. 17:520-527.
Oksbjerg, N., F. Gondret, and M. Vestergaard. 2004. Basic principles of muscle development and growth in meat-producing mammals as affected by the insulin-like growth factor (IGF) system. Domest. Anim. Endocrin. 27:219-240.
Owens, P. C., K. L. Gatford, P. E. Walton, W. Morley, and R. C. Campbell. 1999. The relationship between endogenous insulin-like growth factors and growth in pigs. J. Anim. Sci. 77:2098-2103.
Peng, M., G. Pelletier, M. -F. Palin, S. Véronneau, D. LeBel, and T. Abribat. 1996. Ontogeny of IGFs and IGFBPs mRNA levels and tissue concentrations in liver, kidney and skeletal muscle of pig. Growth Develop. Aging 60:171-187.
Randhawa, R., and P. Cohen. 2005. The role of the insulin-like growth factor system in prenatal growth. Mol. Genet. Metab. 86:84-90.
Rattink, A. P., D. J. de Koning, M. Faivre, B. Harlizius, J. A. M. van Arendonk, and M. A. M. Groenen. 2000. Fine mapping and imprinting analysis for fatness trait QTLs in pig. Mamm. Genome 11:656-661.
Reik, W., and J. Walter. 2001. Genomic imprinting: parental influence on the genome. Nature Reviews 2:21-32.
Roith, D. L. 1997. Insulin-like growth factors. New Engl. J. Med. 27:633-640.

Rust, S., H. Funke, and G. Assmann. 1993. Mutagenically separated PCR (MS-PCR) : a highly specific one step procedure for easy mutation detection. Nucleic Acids Res. 21:3623-3629.
SAS Institute Inc. 2000. SAS/STAT User’s Guide, Version 8. SAS Institute Inc., Cary, NC, USA.
Schlüter, B., M. Erren, H. Schotte, R. Junker, S. Rust, and G. Assmann. 2002. The mutagenically separated polymerase chain reaction is a rapid and reliable method for genotyping of the tumour necrosis factor-α promoter polymorphism (-308G/A). Clin. Chim. Acta 320:135-138.
Schmitz, C., and K. Lindpaintner. 1997. Two specific and simple methods for genotyping of the paraoxonase/arylesterase A/B polymorphism. Genet. Anal-Biomol. E. 14:9-11.
Short, T. H., E. R. Wilson, and D. G. McLaren. 1994. Relationships between growth and litter traits in pig dam lines. Page 413-416 in 5th World Congr. Genet. Appl. Livest. Prod., Guelph, Canada.
Szabó, P. E., S. H. E. Tang, F. J. Silva, W. M. K. Tsark, and J. R. Mann. 2004. Role of CTCF binding sites in the IGF2/H19 imprinting control region. Mol. Cell. Biol. 24:4791-4800.
Tholen, E., K. L. Bunter, S. Hermesch, and H. -U. Graser. 1996. The genetic foundation of fitness and reproduction traits in Australian pig populations: II. Relationships between weaning to conception interval, farrowing interval, stayability and other common reproduction and production traits. Austr. J. Agric. Res. 47:1275-1290.
Tollefsen, S. E., J. L. Sadow, and P. Rotwein. 1989. Coordinate expression of insulin-like growth factor II and its receptor during muscle differentiation. Proc. Natl. Sci. USA 86:1543-1547.
Ulvik, A., J. Ren, H. Refsum, and P. M. Ueland. 1998. Simultaneous determination of methylenetetrahydrofolate reductase C677T and factor V G1691A genotypes by mutagenically separated PCR and multiple-injection capillary electrophoresis. Clin. Biochem. 44:264-269.
Van Laere, A. V., M. Nguyen, M. Braunschweig, C. Nezer, C. Collette, L. Moreau, A. L. Archibald, C. S. Haley, N. Buys, M. Tally, G. Andersson, M. Georges, and L. Andersson. 2003. A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig. Nature 425:832-836.
Vyloulalová, Z., A. Knoll, J. Dvová, and S. Cepica. 2006. New SNPs in the IGF2 gene and association between this gene and backfat thickness and lean meat content in large white pigs. J. Anim. Breed. Genet. 123:204-207.
Zhang, B., H. Tanaka, and K. Saku. 2004. Simple and rapid detection of uncoupling protein-2-866G/A polymorphism by mutagenically separated polymerase chain reaction. Clin. Chim. Acta 344:205-210.