種豬高肉質基因晶片之開發與應用

本研究旨在針對豬隻心臟型脂肪酸結合蛋白(heart fatty acid-binding protein, H-FABP)基因呈現有單一核甘多態性(single nucleotide polymorphisms, SNPs)之特定序列，嘗試開發使之成為高效率篩選具備高肉質遺傳性狀種豬之使用。為謀確認豬隻H-FABP基因之SNPs。試驗首先從已知表現有高肉質性狀之種豬，針對其H-FABP基因多態性片段建立標準序列核酸選殖株，並設定擴增及標定標的基因之多重聚合酶鏈鎖反應系統反應條件。寡聚核苷探針係根據SNPs序列設計並固定於固相支撐物上如：聚己二醯己二胺膜(nylon membrane)上；將PCR擴增子(PCR amplicons) 與探針雜合於晶片上，進行訊號定量及標準化。初步試驗結果首先確定成功建構高專一性之H-FABP寡聚核苷引子並完成單一及多重PCR理想反應條件；藉由20個未知樣品證實每組特異性引子擴增H-FABP基因之SNPs序列時，各自具備高度敏感性與高度特異性；此外為達成多重鑑定SNPs之目的乃進一步設計3組引子已嘗試同步擴增不同基因型，經40頭豬基因組樣品試驗結果，擴增成功率達95%，且所得DNA片段各具備其SNPs基因型。進一步研究則致力於改善以MutS為基礎之蛋白質-核酸混合晶片技術平台建構並測試為此所產生之微陣列檢測系統的靈活性；準確性及其實際應用效果執行能力。試驗首先自E. coli基因選殖配對錯誤結合蛋白MutS核酸序列並將之嵌入PinPoint蛋白質表現系統利於大量表現並純化該MutS融合蛋白。所獲得之MutS融合蛋白功能，經使用六種已知基因型態之H-FABP標準選殖株(AA, aa, DD, dd, HH及 hh 等)，藉由硝化纖維膜上點佈吸漬與前述純化之MutS蛋白進行雜合分析。試驗結果顯示，由H-FABP基因多態性標準選殖株混和製備之異源雙股在理想條件下能夠成功的被MutS重組蛋白所辨識。

關鍵字

基因晶片；心脂肪酸結合蛋白

並列摘要

In this present study, attempts were made to develop an array analysis system for simultaneous genotyping of several single nucleotide polymorphisms (SNPs) located in porcine H-FABP gene and hopefully, an available high throughput screening strategy can be successfully created for selecting the breeding pigs characterized with high performance in meat quality. To meet this purpose, initial studies were first conducted to identify those SNPs found in porcine H-FABP gene. In these initial studies, different genotypes of H-FABP polymorphism fragments from known breeding pigs with elite meat quality were served as standard clones and the oligonucleotide array technology was subjected to setup a multiplexed PCR system suitable for amplification and labeling of the target genes. Oligonucleotide probes were designed according SNPs sequence and were then immobilized on a solid support like nylon membrane. After hybridization of PCR amplicons to probes on a chip, signals were quantified and normalized. From these studies, several results have been obtained, including that of: 1. appropriate probes of oligonucleotides have been successfully designed, according SNPs sequence of H-FABP gene; 2. optimal conditions for both of the single and the multiplexed PCR system have all been successfully verified when a total of 20 unknown samples were subjected for PCR amplification test; 3. experimental results confirmed that each of the specific primer-sets owing characteristics of high sensitivity and high specificity for amplifying and labeling those SNPs sequence against to the H-FABP gene and these are particularly true when multiplexed PCR system were conducted by the combination of 3 or even 4 primer-sets to meet the purpose of multiple identification of SNPs against to each different genotypes at the same time ; and 4. among a total of 40 individual genomic samples tested, over 95 % of them were successfully amplified and each of the DNA fragments obtained were characterized with their specific genotype(s) of SNPs. The subsequent studies were conducted to improve the flexibility, accuracy and executive ability of microarray-based detection system by the use of MutS-based protein-nucleic acid hybrid array technique. Briefly, the mismatch repair binding protein MutS was cloned from E. coli genome and was inserted into PinPoint protein expression system. The fusion protein was expressed and purified. Thereby, the binding efficiency of MutS fusion protein would be easily tested when comparisons were made to those of the H-FABP standard clones. To exam the function of recombinant MutS protein, six types of H-FABP standard clones were mixed with the protein (AA/aa, DD/dd, HH/hh). Those cDNA clones were spotted on nitrocellulose membranes and then hybridize with purified MutS protein. The resulting images will then be available of serving as a quality and quantity control for field studies. Results from these later studies have so far confirmed that three kinds of heteroduplex DNA of H-FABP gene polymorphic standard clones could be discriminated successfully by the recombinant protein MutS when optimal concentrations of heteroduplex DNA and MutS protein had been applied. Further studies conducted to verifying feasibility of these preparations in large scale of field applications are now in progression.

並列關鍵字

gene chip ； heart fatty acid binding protein ； MutS protein

參考文獻

Al Dahouk, S., H. Tomaso, E. Prenger-Berninghoff, W. D. Splettstoesser, H. C. Scholz, and H. Neubauer. 2005.Identification of brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). Crit. Rev. Microbiol. 31(4): 191-196

Allen, D. J., A. Makhov, M. Grilley, J. Taylor, R. Thresher, P. Modrich, and J. D. Griffith. 1997. MutS mediates heteroduplex loop formation by a translocation mechanism. EMBO J. 16: 4467-4476.

Ban, C., and W. Yang. 1998. Structural basis for muth activation in E.coli mismatch repair and relationship of muth to restriction endonucleases. EMBO J. 17: 1526-1534.

Banaszak, L., L. Banaszak, N. Winter, Z. Xu, D. A. Bernlohr, S. Cowan, and T. A. Jones. 1994. Lipid-binding proteins: A family of fatty acid and retinoid transport proteins. Adv. Protein Chem. 45: 89-151.

Behrensdorf, H. A., M. Pignot, N. Windhab, and A. Kappel. 2000. Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format. Nucl. Acids Res. 30: E64.

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種豬高肉質基因晶片之開發與應用

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