Determination of intronic sequences adjacent to an exon using polymerase chain reaction and genomic DNA library constructed by TA cloning.
Determination of intronic sequences adjacent to an exon using polymerase chain reaction and genomic DNA library constructed by TA cloning.
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使用聚合酶链式反应和通过 TA 克隆构建的基因组 DNA 文库确定与外显子相邻的内含子序列。
DOI：
10.1006/abio.2000.4897
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发表时间：
2001
期刊：
影响因子：
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通讯作者：
Riddell,SR
中科院分区：
文献类型：
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作者：
Akatsuka,Y;Warren,EH;Brickner,AG;Engelhard,VH;Riddell,SR
确定外显子侧翼的内含子 DNA 片段序列对于设计跨越完整外显子的 PCR 引物或分析剪接供体/受体结构通常至关重要。 Cosmid 文库筛选、反向 PCR (1–3)、Alu PCR (4) 和 Panhandle PCR (5) 均已用于获取位于已知序列边界之外的 DNA 序列。当需要分析完整的基因组结构时，粘粒文库最有用；然而，DNA文库的构建和筛选以及重复测序是很麻烦的。反向 PCR (1-3) 因其可行性而被广泛使用，但该方法需要成功的自身（或分子内）连接，然后使用位于已知外显子序列中的引物进行 PCR 扩增。反向PCR存在几个问题，包括难以环化短于200-300 bp的片段、难以扩增大的环化目标片段（同时会产生复杂的异质片段）以及分子间连接的发生，导致序列包含不相关片段。为了克服这些问题，有必要优化反应，例如在环化和 PCR 中使用较低浓度的 DNA。 Panhandle PCR 对于确定内含子序列也有效，但需要优化和许多步骤 (5)。我们最近在 KIAA0020 基因的外显子中发现了多态性，该基因编码次要组织相容性抗原 (mHA) 2，参与同种异体干细胞移植后的移植物抗宿主反应（8，AG Brickner，准备中的手稿）。确定 KIAA0020 5 侧翼内含子序列对于设计用于基因分型供体和受体的等位基因特异性引物是必要的，因为从 cDNA 序列设计的有义引物序列被长内含子打断，即使使用长 PCR 技术也无法扩增。传统的反向 PCR 方法也无法扩增该内含子序列。最近，TA 克隆被描述为构建基因组 DNA 文库的有效方法 (6, 7)。我们在这里报告使用这种方法结合 PCR 来获得 KIAA0020 基因外显子侧翼的内含子 DNA 序列。
The determination of the sequence of intronic DNA segments flanking exons is often crucial for designing PCR primers that will span complete exons or for the analysis of splice donor/acceptor structure. Cosmid library screening, inverse PCR (1–3), Alu PCR (4), and Panhandle PCR (5) have all been used to obtain the sequence of DNA that lies outside the boundaries of a known sequence. A cosmid library is most useful when complete genomic structure needs to be analyzed; however, construction and screening of DNA libraries followed by repeated sequencing is cumbersome. Inverse PCR (1–3) has been widely used because of its feasibility but this method requires successful self (or intramolecular)-ligation followed by amplification by PCR with primers located in the known exonic sequences. There are several problems with inverse PCR including the difficulty circularizing fragments shorter than 200–300 bp, difficulty amplifying large circularized target fragments where complex heterogeneous fragments are also generated, and the occurrence of intermolecular ligations which result in sequences containing irrelevant fragments. To overcome these problems, it is necessary to optimize the reactions such as by using a lower concentration of DNA in circularization and PCR. Panhandle PCR is also effective for determining intron sequence but requires optimization and many steps (5).We have recently identified a polymorphism in an exon of the KIAA0020 gene that encodes a minor histocompatibility antigen (mHA) 2 involved in graft versus host reactions after allogeneic stem cell transplant (8, AG Brickner, manuscript in preparation). The determination of the 5-flanking intron sequence of KIAA0020 was necessary for designing allele-specific primers for genotyping donors and recipients because the sense primer sequence designed from the cDNA sequence was interrupted by a long intron, which could not be amplified even with long PCR techniques. A conventional inverse PCR approach also failed to amplify this intronic sequence. Recently, TA cloning has been described as an efficient method for construction of genomic DNA libraries (6, 7). We report here the use of this method combined with PCR for obtaining the sequence of intronic DNA flanking an exon of the KIAA0020 gene.