Key laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, PR China.
BMC Biotechnol. 2011 Nov 17;11:109. doi: 10.1186/1472-6750-11-109.
The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow.
Here, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR) for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs). These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis.
The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures.
基于基因组学的技术的出现彻底改变了许多生物研究领域。然而，染色体步移或侧翼序列克隆仍然是确定基因结构的必要和重要程序。这些方法用于确定 T-DNA 插入位点，因此对于已经创建了大型 T-DNA 插入文库的生物体（如水稻和拟南芥）特别相关。目前用于侧翼序列克隆的方法，包括流行的 TAIL-PCR 技术，相对来说比较繁琐和缓慢。
在这里，我们报告了一种简单有效的融合引物和嵌套集成 PCR 方法（FPNI-PCR），用于鉴定和克隆已知序列侧翼的未知基因组区域。简而言之，设计了一组通用引物，由各种 15-16 个碱基的任意简并寡核苷酸组成。这些任意简并引物融合到一个接头寡核苷酸的 3' 端，该接头寡核苷酸提供了一个没有简并核苷酸的已知序列，从而形成融合引物（FPs）。这些融合引物用于集成嵌套 PCR 策略的第一步，该策略定义了整个 FPNI-PCR 方案。为了证明这种新策略的有效性，我们已经成功地使用它分离了多个基因组序列，即各种蔷薇科物种中的 21 个同源基因、玫瑰 rugosa 的 4 个 MYB 基因、矮牵牛杂交种转录因子的 3 个启动子，以及转基因烟草品系中的 4 个 T-DNA 插入位点侧翼序列和水稻和拟南芥测序基因组中的 6 个特定基因。
通过 FPNI-PCR 成功扩增靶产物验证了这种新策略是一种有效、低成本且简单的方法。此外，FPNI-PCR 比已建立的 TAIL-PCR 和 hiTAIL-PCR 程序更敏感、快速和准确。
