Qin L, Prins P, Jones J T, Popeijus H, Smant G, Bakker J, Helder J
The Graduate School for Experimental Plant Sciences, Laboratory of Nematology, Wageningen University and Research Center, Binnenhaven 10, 6709 PD Wageningen, The Netherlands.
Nucleic Acids Res. 2001 Apr 1;29(7):1616-22. doi: 10.1093/nar/29.7.1616.
The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of approximately 4000 genes in five developmental stages produced by cDNA-AFLP.
大规模基因组和表达序列标签（EST）项目所释放的大量DNA序列数据凸显了开发高效且廉价方法的必要性，以便将序列数据库与时空表达谱相联系。在此，我们展示了将cDNA序列数据（包括EST序列）与cDNA-AFLP所揭示的转录本谱相联系的强大功能，cDNA-AFLP是一种基于限制性酶切消化和高严格条件下选择性扩增的高度可重复的差异显示方法。我们开发了一个计算机程序（GenEST），它可以预测从数据库中检索到的电子消化cDNA序列的虚拟转录本衍生片段（TDF）的大小。绝大多数产生的虚拟TDF可以在cDNA-AFLP凝胶上显示的数千个TDF中找到踪迹。对从cDNA-AFLP凝胶上切下的相应条带进行测序，结果没有发现不一致之处。因此，可以非常有效地筛选cDNA序列数据库，以识别具有相关表达谱的基因。反之，也可以从cDNA-AFLP凝胶切换到数据库中的序列。利用限制性酶识别位点、引物延伸和估计的TDF大小作为标识符，可以识别与具有有趣表达模式的TDF相对应的DNA序列。在本文中，我们通过分析植物寄生线虫罗斯托克根结线虫展示了两个方向的实例。通过将感染期幼虫的EST与cDNA-AFLP产生的五个发育阶段中约4000个基因的表达谱相结合，鉴定出了各种新的致病因子。
