High throughput functional screening of cDNAs

Details High throughput functional screening of cDNAs High throughput functional screening of cDNAs

1999-12-03

2001-08-14

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S024300, C536S024310, C536S024320, C536S024330, C536S025300, C536S025320

Reexamination Certificate

active

06274321

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to functional genomics, and more particularly to high throughput screening of cDNAs expressing products that interact with target molecules of interest.
The initial “structural” phases of the various genome projects have made rapid progress in mapping and sequencing genes. In the next phase, “functional genomics” will explore the functions of these newly identified genes, so their gene products can be identified and characterized by their interactions with other molecules. This will allow genes and their products to be understood in the context of their roles in metabolic pathways, cell-signaling and other complex systems.
Previously, such interactions could only be characterized by laborious and technically demanding biochemical purification to identify the protein of interest, followed by amino acid sequencing and cloning of the gene encoding the identified protein.
Recently, the yeast two-hybrid system has been used, but it can be tedious to perform. The system can also result in a high number of false positives, often caused by factors within the yeast cells, which are not easily controlled. Phage-display libraries have also been used with varying degrees of success. Nevertheless, neither method can be used to readily identify cDNAs encoding proteins that interact with a target molecule of interest.
A further difficulty is that manually sifting through large numbers of genes can be an overwhelming task, even when represented by cDNA clones. Moreover, the products of some of the most functionally important genes such as regulatory genes may be in low abundance, have weak interactions or may participate in molecular interactions that are difficult to manipulate in complex mixtures or in vivo. As a result, throughput is low and assay sensitivity is dependent on the strength of the interaction and complexity of the mixture.
Thus, there is a need for rapid and high throughput functional screening of cDNA libraries. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides methods for screening cDNA libraries or arrays of known cDNAs to identify individual cDNAs that express a product having an interaction with a target molecule. First, individual cDNAs are pooled and the pools are expressed, for example by using in vitro transcription/translation. The expression products are then assayed for interaction with a selected target molecule, using an assay such as a scintillation proximity assay (SPA). cDNAs associated with expression pools of interest are selectively re-pooled using a different pooling scheme. By repeating the expression and assay steps for the re-pooled cDNAs, an individual cDNA can be rapidly identified.
The screening method is readily automated in a computer-controlled device for high throughput screening, controlled by software for performing the screening method. The invention also provides methods of transfecting a cell with a cDNA identified by the screening method to confer a desired property to a cell.


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