Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning.
Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning.
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融合引物和巢式整合PCR（FPNI-PCR）：快速染色体步移或侧翼序列克隆的高效新策略
DOI：
10.1186/1472-6750-11-109
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发表时间：
2011-11-17
影响因子：
3.5
通讯作者：
Ning G
中科院分区：
文献类型：
--
作者：
Wang Z;Ye S;Li J;Zheng B;Bao M;Ning G
背景基于基因组学的技术的出现彻底改变了生物学研究的许多领域。然而，染色体行走或侧翼序列克隆仍然是确定基因结构的必要且重要的程序。此类方法用于识别 T-DNA 插入位点，因此对于已创建大型 T-DNA 插入文库的生物体尤其相关，例如水稻和拟南芥。目前可用的侧翼序列克隆方法，包括流行的TAIL-PCR技术，相对费力且缓慢。结果在这里，我们报告了一种简单有效的融合引物和巢式集成PCR方法（FPNI-PCR），用于识别和克隆已知序列侧翼的未知基因组区域。简而言之，设计了一组由各种15-16碱基任意简并寡核苷酸组成的通用引物。将这些任意简并引物融合至接头寡核苷酸的3'端，该接头寡核苷酸提供已知序列而没有简并核苷酸，从而形成融合引物(FP)。这些融合引物用于定义整体 FPNI-PCR 协议的集成巢式 PCR 策略的第一步。为了证明这一新策略的有效性，我们成功地使用它分离了多个基因组序列，即蔷薇科各物种中的21个基因直向同源物、蔷薇的4个MYB基因、矮牵牛的3个转录因子启动子、转基因烟草品系中的4个T-DNA插入位点侧翼序列和来自已测序的水稻基因组的6个特定基因 结论通过FPNI-PCR对目标产物的成功扩增证实了该新策略是一种有效、低成本且简单的方法。此外，FPNI-PCR 代表了比已建立的 TAIL-PCR 和 hi TAIL-PCR 程序更灵敏、快速和准确的技术。
BackgroundThe 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 andArabidopsis. The currently available methods for flanking sequence cloning, including the popularTAIL-PCRtechnique, are relatively laborious and slow.ResultsHere, 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 overallFPNI-PCRprotocol. 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, 4MYBgenes 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 andArabidopsis.ConclusionsThe successful amplification of target products throughFPNI-PCRverified that this novel strategy is an effective, low cost and simple procedure. Furthermore,FPNI-PCRrepresents a more sensitive, rapid and accurate technique than the establishedTAIL-PCRandhiTAIL-PCRprocedures.
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影响因子：
3.7
作者：
Ji J;Braam J
通讯作者：
Braam J
一种新颖且简单的 PCR 步移方法，可快速获取微生物基因组中已知位点侧翼的长 DNA 序列
影响因子：
2.6
作者：
Luo, Peng;Su, Ting;Ren, Chunhua
通讯作者：
Ren, Chunhua
影响因子：
4.4
作者：
Wang, Shiming;He, Jian;Li, Shunpeng
通讯作者：
Li, Shunpeng
影响因子：
14.9
作者：
Yan, YX;An, CC;Chen, ZL
通讯作者：
Chen, ZL
影响因子：
2
作者：
He, Y. H.;Ning, G. G.;Bao, M. Z.
通讯作者：
Bao, M. Z.