Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification
Generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification
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DOI：
10.1073/pnas.97.1.349
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发表时间：
2000-01-04
影响因子：
11.1
通讯作者：
Wang, SM
中科院分区：
文献类型：
--
作者：
Chen, JJ;Rowley, JD;Wang, SM
我们已经开发了一种称为从用于基因鉴定（GLGI）的基因表达序列分析（SAGE）标签产生较长cDNA片段的技术，以将10个碱基的SAGE标签转化为其相应的覆盖数百个碱基的3’cDNA片段。在PCR中，使用含有10个碱基的SAGE标签的引物作为有义引物，并且使用单碱基锚定的寡核苷酸（dT）引物作为反义引物，与Pfu DNA聚合酶一起。通过使用这种方法，可以产生从SAGE标签向相应序列的3'末端延伸的cDNA片段。GLGI技术的应用可以解决SAGE技术应用中的两个关键问题：一是可以产生与SAGE标签相对应的较长片段，该片段在数据库中没有匹配，用于进一步研究;二是可以从与相同SAGE标签匹配的多个序列中鉴定出与SAGE标签相对应的特定片段。GLGI方法的发展提供了几个潜在的应用。首先，它提供了一个战略，甚至更广泛的应用SAGE技术的定量分析的全球基因表达。第二，SAGE/GLGI的联合应用可用于完成人类和其他真核生物表达基因的编目。第三，它可用于鉴定来自基因内任何外显子的3' cDNA序列。它也可以用于确认基因组序列中生物信息学工具预测的外显子的真实性。第四，SAGE/GLGI的组合应用可以应用于确定人类和其他真核生物基因组中基因组序列中表达基因的3'边界。
We have developed a technique called the generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI), to convert SAGE tags of 10 bases into their corresponding 3' cDNA fragments covering hundred bases, A primer containing the 10-base SAGE tag is used as the sense primer, and a single base anchored oligo(dT) primer is used as an antisense primer in PCR, together with Pfu DNA polymerase. By using this approach, a cDNA fragment extending from the SAGE tag toward the 3' end of the corresponding sequence can be generated. Application of the GLGI technique can solve two critical issues in applying the SAGE technique: one is that a longer fragment corresponding to a SAGE tag, which has no match in databases, can be generated for further studies; the other is that the specific fragment corresponding to a SAGE tag can be identified from multiple sequences that match the same SAGE tag. The development of the GLGI method provides several potential applications. First, it provides a strategy for even wider application of the SAGE technique for quantitative analysis of global gene expression. Second, a combined application of SAGE/GLGI can be used to complete the catalogue of the expressed genes in human and in other eukaryotic species. Third, it can be used to identify the 3' cDNA sequence from any exon within a gene. It can also be used to confirm the reality of exons predicted by bioinformatic tools in genomic sequences. Fourth, a combined application of SAGE/GLGI can be applied to define the 3' boundary of expressed genes in the genomic sequences in human and in other eukaryotic genomes.