TAIL-PCR研究进展及其在畜牧兽医中的应用

发布时间：2018-01-23 07:56

本文关键词： TAIL-PCR 染色体步移研究进展应用　出处：《南方农业学报》2017年05期 　论文类型：期刊论文

【摘要】：热不对称交错PCR(TAIL-PCR)是一种以PCR为基础的染色体步移,主要用于分离已知基因的侧翼序列,具有简便、快速、高效、特异性强等特点。文章从TAIL-PCR的原理、优势及局限性、反应体系优化等方面对TAIL-PCR的研究进展进行综述,其中在畜牧兽医中主要用于已知基因侧翼克隆和转基因动物模型外源基因插入位点确定。但由于基因组DNA的庞大和复杂性,使得从基因组中克隆获得目的基因仍存在诸多限制因素,如特异性引物错配和随机引物结合位点不确定,易造成非特异性产物的产生;模板GC含量异常或存在高度重复序列时,即使使用多种简并引物也难以获得阳性产物。因此,应根据实际情况对TAIL-PCR进行灵活调整。当模板较复杂时,可借鉴连接介导PCR原理,给引物加上接头,提高扩增反应的特异性;当模板较简单或序列较清晰时,可根据模板特点(如高GC含量)或已知模板序列设计引物、调整反应条件,增加操作的简便性;还可参考Suppression-PCR原理,选择引物和反应条件,获得较长的扩增片段。此外,TAIL-PCR可与多种技术联合使用,促使其在畜牧兽医中获得更广泛深入的应用。
[Abstract]:Thermal asymmetric staggered PCR (TAIL-PCR) is a kind of chromosome step based on PCR, which is mainly used to isolate the flanking sequence of known genes. It is simple, fast and efficient. The principle, advantages and limitations of TAIL-PCR and the optimization of reaction system are reviewed in this paper. It is mainly used in animal husbandry and veterinarian for the cloning of known gene flanking and the determination of foreign gene insertion site in transgenic animal model. But because of the large and complex genomic DNA. There are still many limiting factors for cloning and obtaining target genes from genomes, such as mismatch of specific primers and uncertainty of random primer binding sites, which can easily lead to the production of non-specific products. When the GC content of template is abnormal or there is a high repeat sequence, it is difficult to obtain positive products even if multiple degenerate primers are used. The TAIL-PCR should be adjusted flexibly according to the actual situation. When the template is more complex, we can use the principle of ligation mediated PCR and add the primers with the connector to improve the specificity of the amplification reaction. When the template is simple or the sequence is clear, primers can be designed according to the characteristics of template (such as high GC content) or known template sequence to adjust the reaction conditions and increase the simplicity of operation. According to the principle of Suppression-PCR, we can select primers and reaction conditions to obtain longer amplified fragments. In addition, TAIL-PCR can be used in combination with many techniques. It promotes its wider and deeper application in animal husbandry and veterinary medicine.
【作者单位】：广西兽医研究所/广西动物疫病病原生物学与诊断重点实验室;亚热带农业生物资源保护与利用国家重点实验室;广西大学生命科学与技术学院;
【基金】：国家自然科学基金项目(31260613) 广西自然科学基金项目(2013GXNSFBA019123) 广西畜禽疫苗新技术重点实验室开放基金项目(14-045-31-B-5)
【分类号】：Q78;S85
【正文快照】： 0引言获得较完整基因序列是研究基因结构、功能和表达调控的必要条件,确定外源基因的插入位点是建立转基因动植物模型和插入突变体后的首要步骤,掌握反转录病毒科病毒在宿主基因组中的整合位点是研究病毒致病机理的根本前提,而开展这些研究均需要从已知基因序列出发,通过染色

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