高效TAIL-PCR方法.docVIP

扩增已知序列旁邻的未知DNA序列的高效TAIL-PCR方法High-efficient thermal asymmetric interlaced PCR （hiTAIL-PCR）Want to mining unknown sequences quickly?Just hi-TAIL it!引子: 扩增已知序列旁邻的未知DNA序列是分子生物学研究中非常重要的任务。华南农业大学的Yao-Guang Liu 教授发明的TAIL-PCR是解决该问题的有效方法。为了提高获得特异的、长片段目标产物的成功率，最近刘教授对TAIL-PCR方法作了较大改进，创建了新的hiTAIL-PCR方法（Liu and Chen，2007）。该方法用在水稻、拟南芥、番木瓜、昆虫等生物的未知DNA序列扩增都获得很好效果。本文所介绍LAD简并引物及AC1引物都是通用的，而特异引物序列是基于CAMBIA载体1302、1305、1300的RB边界附近序列设计的，用于扩增上述载体的转基因植株的T-DNA插入点旁邻未知基因组序列非常有效。使用者也可根据本文的原理，利用本文的LAD简并引物及AC1引物，再加上自行设计的特异引物，扩增其他类型的未知DNA序列。参考文献：High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Yao-Guang Liu and Yuanling Chen. BioTechniques Vol. 43, No. 5: pp 649-656 (Nov 2007)AbstractIsolation of unknown DNA sequences flanked by known sequences is an important task in molecular biology research. Thermal asymmetric interlaced PCR (TAIL-PCR) is an effective method for this purpose. However, the success rate of the original TAIL-PCR needs to be increased, and it is more desirable to obtain target products with larger sizes. Here we present a substantially improved TAIL-PCR procedure with special primer design and optimized thermal conditions. This high-efficiency TAIL-PCR (hiTAIL-PCR) combines the advantages of the TAIL-cycling and suppression-PCR, thus it can block the amplification of nontarget products and suppress small target ones, but allow efficient amplification of large target sequences. Using this method, we isolated genomic flanking sequences of T-DNA insertions from transgenic rice lines. In our tests, the success rates of the reactions were higher than 90%, and in most cases the obtained major products had sizes of 1–3 kb.该杂志是免费登陆浏览的：下面介绍一下该方法的原理及设计引物和实验过程应该注意的事项，供参考。如在使用该方法过程中有信息或问题需反馈，可直接留言，以便改进。1. TAIL-PCR 1.1 原理 A 利用简并引物在靶序列上创造结合位点B 利用nested PCR提高扩增产物特异性C 利用2轮高温、1轮低温交替的超级PCR循环提高特异产物的扩增，降低非特异产物的比例D 模板高倍数稀释以降低非特异产物在下一轮作模板的比例E 通过电泳检测第2轮和第3轮产物相差的大小来判断产物的特异性图1: 第一轮扩增图2: 第二轮扩增图3: 第三