Orcheski Benjamin B, Davis Thomas M
Department of Biological Sciences, University of New Hampshire, Durham, NH, 03824, USA.
BMC Res Notes. 2010 Mar 4;3:61. doi: 10.1186/1756-0500-3-61.
Although technological advances allow for the economical acquisition of whole genome sequences, many organisms' genomes remain unsequenced, and fully sequenced genomes may contain gaps. Researchers reliant upon partial genomic or heterologous sequence information require methods for obtaining unknown sequences from loci of interest. Various PCR based techniques are available for sequence walking - i.e., the acquisition of unknown DNA sequence adjacent to known sequence. Many such methods require rigid, elaborate protocols and/or impose narrowly confined options in the choice of restriction enzymes for necessary genomic digests. We describe a new method, TOPO(R) Vector-Ligation PCR (or TVL-PCR) that innovatively integrates available tools and familiar concepts to offer advantages as a means of both targeted sequence walking and paralog mining.
TVL-PCR exploits the ligation efficiency of the pCR(R)4-TOPO(R) (Invitrogen, Carlsbad, California) vector system to capture fragments of unknown sequence by creating chimeric molecules containing defined priming sites at both ends. Initially, restriction enzyme-digested genomic DNA is end-repaired to create 3' adenosine overhangs and is then ligated to pCR4-TOPO vectors. The ligation product pool is used directly as a template for nested PCR, using specific primers to target orthologous sequences, or degenerate primers to enable capture of paralogous gene family members. We demonstrated the efficacy of this method by capturing entire coding and partial promoter sequences of several strawberry Superman-like genes.
TVL-PCR is a convenient and efficient method for DNA sequence walking and paralog mining that is applicable to any organism for which relevant DNA sequence is available as a basis for primer design.
尽管技术进步使得经济地获取全基因组序列成为可能，但许多生物体的基因组仍未测序，而且已完全测序的基因组可能存在缺口。依赖部分基因组或异源序列信息的研究人员需要从感兴趣的基因座获取未知序列的方法。各种基于PCR的技术可用于序列步移，即获取与已知序列相邻的未知DNA序列。许多此类方法需要严格、复杂的方案，并且在选择用于必要基因组消化的限制酶时提供的选择范围很窄。我们描述了一种新方法，TOPO(R)载体连接PCR（或TVL-PCR），它创新性地整合了现有工具和常见概念，作为一种靶向序列步移和旁系同源物挖掘的手段具有优势。
TVL-PCR利用pCR(R)4-TOPO(R)（Invitrogen，加利福尼亚州卡尔斯巴德）载体系统的连接效率，通过创建两端含有确定引物位点的嵌合分子来捕获未知序列的片段。最初，用限制酶消化的基因组DNA进行末端修复以产生3'腺苷突出端，然后与pCR4-TOPO载体连接。连接产物池直接用作巢式PCR的模板，使用特异性引物靶向直系同源序列，或使用简并引物捕获旁系同源基因家族成员。我们通过捕获几个草莓类超人基因的完整编码序列和部分启动子序列证明了该方法的有效性。
TVL-PCR是一种方便、高效的DNA序列步移和旁系同源物挖掘方法，适用于任何有相关DNA序列可作为引物设计基础的生物体。
