Development and application of gene chips designed for screening of breeding pigs with high performance in meat quality
|Keywords:||基因晶片;心脂肪酸結合蛋白;gene chip;heart fatty acid binding protein;MutS protein||Issue Date:||2006||Abstract:||
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
聚己二醯己二胺膜(nylon membrane)上；將PCR擴增子(PCR amplicons)
用六種已知基因型態之H-FABP標準選殖株(AA, aa, DD, dd, HH及 hh
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.
|Appears in Collections:||動物科學技術學系|
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