Du Zeji, Scott Angela D, May Gregory D
Samuel Roberts Noble Foundation, Ardmore, OK, USA.
Biotechniques. 2003 Jul;35(1):66-7, 70-2. doi: 10.2144/03351st01.
As a method for accurate quantitative and qualitative analysis of cell transcription, serial analysis of gene expression (SAGE) has been widely used in biological, medical, and pharmaceutical areas of research. The numerous steps required in the SAGE protocol, however, limit the efficiency of SAGE library construction. PCR amplification of ditags and concatemer separation are two such steps. Primer-2, which was used to amplify ditags in the original SAGE protocol, was prone to self-anneal and resulted in a decreased efficiency of the PCR amplification step. We have designed a new primer pair, which substantially enhances the efficiency of PCR amplification. Incorporating this modification reduces the number of reactions required for SAGE library construction. Sixty 50-microL reactions are now sufficient for SAGE library construction (up to an 80% reduction in comparison to the original protocol). Concatemer separation by column filtration and subsequent gel separation has been modified and has proven to be more efficient than simple heat treatment of the ligation mixture. Both of the above modifications may also be suitable for the newly developed LongSAGE protocol.
作为一种用于细胞转录精确的定量和定性分析的方法，基因表达系列分析（SAGE）已广泛应用于生物学、医学和药学研究领域。然而，SAGE方案所需的众多步骤限制了SAGE文库构建的效率。双标签的PCR扩增和多联体分离就是这样的两个步骤。在原始SAGE方案中用于扩增双标签的引物-2容易发生自身退火，导致PCR扩增步骤的效率降低。我们设计了一对新引物，可大幅提高PCR扩增的效率。纳入此修改可减少构建SAGE文库所需的反应数量。现在60个50微升反应就足以构建SAGE文库（与原始方案相比减少了多达80%）。通过柱过滤和随后的凝胶分离进行多联体分离已得到改进，并且已证明比简单地对连接混合物进行热处理更有效。上述两种修改也可能适用于新开发的LongSAGE方案。
