TAIL-PCR研究进展及其在畜牧兽医中的应用

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。

Advances in TAIL-PCR and its application in animal husbandry and veterinary

Simple, rapid, high efficient and specific, thermal asymmetric interlaced PCR(TAIL-PCR), a kind of chromosome walking based on PCR, was mainly used to isolate the flanking sequences of target DNA segments. The princi-ple, advantage, disadvantage and reaction system optimization of TAIL-PCR as well as some critical problem were summa-rized. In animal husbandry and veterinary medicine, TAIL-PCR was 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 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 templates 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 (high GC 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 Suppression-PCR. Moreover , TAIL-PCR could be used with other technologies to facilitate its application in animal husbandry and veterinary.