Jia Xianbo, Lin Xinjian, Chen Jichen
Institute of Soil and Fertilizer, Fujian Academy of Agricultural and Sciences, Wusi Road, Fuzhou, 350001, Fujian, People's Republic of China.
Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou, People's Republic of China.
AMB Express. 2017 Nov 2;7(1):195. doi: 10.1186/s13568-017-0495-x.
Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.
目前的基因组步移方法非常耗时，而且许多方法会产生非特异性扩增产物。为了扩增粘质沙雷氏菌FZSF02中与Tn5转座子插入位点相邻的侧翼序列，我们开发了一种基于热不对称交错PCR（TAIL-PCR）的基因组步移方法。这种PCR方法在指数扩增步骤之前增加了一个20个循环的线性扩增步骤，以提高目标序列的浓度。将线性扩增和指数扩增的产物稀释100倍，以降低导致非特异性扩增的模板浓度。该方法使用了具有高延伸速度的快速DNA聚合酶，并采用一个扩增程序来快速扩增长的特异性序列。通过这种线性和指数热不对称交错PCR（LETAIL-PCR），我们在3小时内成功地从Tn5转座子插入突变菌株中获得了大于2 kb的产物。该方法可广泛应用于基因组步移研究，以扩增与已知序列相邻的未知序列。
