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    • 6. 发明申请
    • COLONY ARRAY-BASED cDNA LIBRARY NORMALIZATION BY HYBRIDIZATIONS OF COMPLEX RNA PROBES AND GENE SPECIFIC PROBES
    • 通过复合RNA探针和基因特异性探针杂交的基于阵列的cDNA文库正常化
    • WO2002095072A1
    • 2002-11-28
    • PCT/US2002/015113
    • 2002-05-10
    • LARGE SCALE BIOLOGY CORPORATIONWEI, Chia-LinRUAN, YijunZHENG, Wenjiin
    • WEI, Chia-LinRUAN, YijunZHENG, Wenjiin
    • C12Q1/68
    • C12N15/1096
    • Each cell normally has a widely differing number of mRNA transcribed for each gene. Consequently, a fill-length cDNA library constructed from the mRNA would also have a widely differing number of cDNA for each gene. A normalized library of the full-length cDNA of a cell is usful for basic, applied, industrial, and medical research. This invention provides for a method for constructing a normalized full-length cDNA library by probing the members of a non-normalized cDNA library with a library of probes generated from mRNA in order to identigy the cDNA of genes that have low or high expression. A collection of the cDNA from the library of the genes that have low expression would constitute a normalized library of these genes. This invention also provided for a method to reduce the number cDNA of genes that have high expression represented by probing these cDNA with a library of probes generated from a small randomly selected number of these cDNA. cDNA that hybridize are represented within this small randomly selectes number of cDNA, while cDNA that do not hybridize are not represented. The latter cDNA can undergo further such probing to further reduce the number of cDNA represented. The cDNA from the library of the genes that have low expression and the randomly selected highly expressed cDNA would constitute a normalized library of these genes.
    • 每个细胞通常具有每个基因转录的mRNA数量差异很大。 因此,由mRNA构建的填充长度cDNA文库对于每个基因也将具有大量不同数量的cDNA。 细胞全长cDNA的归一化库对于基础,应用,工业和医学研究是有用的。 本发明提供了通过用mRNA产生的探针文库探测非标准化cDNA文库的成员来构建归一化全长cDNA文库的方法,以便鉴定具有低表达或高表达的基因的cDNA。 来自具有低表达的基因的文库的cDNA的集合将构成这些基因的归一化文库。 本发明还提供了通过用随机选择数量小的这些cDNA产生的探针文库探索这些cDNA来减少具有高表达性的基因的cDNA数量的方法。 在该小的随机选择数量的cDNA中表达杂交的cDNA,而不表示不杂交的cDNA。 后一种cDNA可以进一步进行这种探测,以进一步减少所表达的cDNA的数量。 来自低表达基因的文库和随机选择的高表达cDNA的cDNA将构成这些基因的归一化文库。