TAIL-PCR研究进展及其在畜牧兽医中的应用

1、TAIL-PCR研究进展及其在畜牧兽医中的应用打开文本图片集摘要：熟不对称交错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

2、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。关键词：TAIL-PCR ;染色体步移；研究进展；应用中图分类号：Q813文献标志码:A文章编号:2095-1191 ( 2017 ) 05-0

3、926-07Advances in TAIL-PCR and its application in animalhusbandry and veterinaryMA Ling 1 , WANG Zhi-qiang 2,3, QIN Shao-minl , WU Jian-minl (1 Guangxi Veterinary Research Institute / Guangxi Key Laboratory of Animal Epidemic Etiology and Diagnostic , Nanning 530001 , China ; 2 State Key Laboratory

4、for Conservation and Utilization of Subtropical Agro-Bioresearches , Nanning 530004 , China ; 3 College of Life Science and Technology , Guangxi University , Nanning 530004 , China )Abstract : Simple , rapid , high efficient and specific , thermal asymmetric interlaced PCR ( TAIL-PCR ), a kind of ch

5、romosome walking based on PCR , was mainly used to isolate the flanking sequences of target DNA segments. The principle , advantage , disadvantage and reaction system optimization of TAIL-PCR as well as some critical problem were summarized. In animal husbandry and veterinary medicine , TAIL-PCR was

6、 mainly used to isolate exogenous gene insertion sites and clone flanking sequences of the known genes in transgenic animals. Being limited by the complexity of genome DNA , there were so many limited factors preventing cloning target genes from genome , such as amplification of nonspecific products

7、 caused by mismatch of specific primers and arbitrary primers , and the difficulties in obtaining positive products even using multiple degenerate primers caused by abnormal template GC content and highly repeated sequences. So the adjustments should be made depend on actual situation. When the temp

8、lates were complex , adapter could be added to the primers to improve the specificity based on the principle of LM-PCR ; while when the template were simple or the sequence were clear, primers could be designed and reaction conditions could be set based on the characters of the templates( highGC content) or known template sequence , which made it easier to operate ; and the suitable primers and reaction conditions could be chosen to get longer amplification products based on the principle of Suppressi