Dias Neto E, Harrop R, Correa-Oliveira R, Wilson R A, Pena S D, Simpson A J
Centro de Pesquisas René Rachou-FIOCRUZ, Belo Horizonte, Brazil.
Gene. 1997 Feb 20;186(1):135-42. doi: 10.1016/s0378-1119(96)00699-3.
The generation of expressed sequenced tags (ESTs) depends on the arbitrary selection of individual cDNA clones from libraries. The efficiency of this process reflects the clonal structure of the library used and can be significantly increased using size selected, directional, normalized cDNA libraries. This strategy, however, is not readily applicable when mRNA is limiting, as is the case in the study of complex microorganisms such as parasites, fetal tissues or tumor biopsies. We show here that the construction and systematic sequencing of minilibraries of cDNAs produced by arbitrarily primed PCR provides an alternative means of efficiently generating ESTs in situations where only nanogram quantities of RNA are available. This methodology greatly compensates for unequal message abundance, avoids the need for complex library construction, is equally applicable to the analysis of abundant or rare biological material and is ideally suited to multicenter programmes.
表达序列标签（EST）的产生依赖于从文库中随机挑选单个cDNA克隆。这个过程的效率反映了所用文库的克隆结构，使用大小选择、定向、标准化的cDNA文库可显著提高效率。然而，当mRNA有限时，如在寄生虫、胎儿组织或肿瘤活检等复杂微生物研究中，这种策略就不太适用了。我们在此表明，通过任意引物PCR产生的cDNA微型文库的构建和系统测序，为在仅有纳克量RNA可用的情况下高效产生EST提供了一种替代方法。这种方法极大地弥补了信息丰度的不均等，避免了复杂文库构建的需要，同样适用于丰富或稀有生物材料的分析，并且非常适合多中心项目。
