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Inverse drug screens: a rapid and inexpensive method for implicating molecular targets.

ABSTRACT: Identification of gene products that function in some specific process of interest is a common goal in developmental biology. Although use of drug compounds to probe biological systems has a very long history in teratology and toxicology, systematic hierarchical drug screening has not been capitalized upon by the developmental biology community. This "chemical genetics" approach can greatly benefit the study of embryonic and regenerative systems, and we have formalized a strategy for using known pharmacological compounds to implicate specific molecular candidates in any chosen biological phenomenon. Taking advantage of a hierarchical structure that can be imposed on drug reagents in a number of fields such as ion transport, neurotransmitter function, metabolism, and cytoskeleton, any assay can be carried out as a binary search algorithm. This inverse drug screen methodology is much more efficient than exhaustive testing of large numbers of drugs, and reveals the identity of a manageable number of specific molecular candidates that can then be validated and targeted using more expensive and specific molecular reagents. Here, we describe the process of this loss-of-function screen and illustrate its use in uncovering novel bioelectrical and serotonergic mechanisms in embryonic patterning. This technique is an inexpensive and rapid complement to existing molecular screening strategies. Moreover, it is applicable to maternal proteins, and model species in which traditional genetic screens are not feasible, significantly extending the opportunities to identify key endogenous players in biological processes.

SUBMITTER: Adams DS

PROVIDER: S-EPMC3142945 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Inverse drug screens: a rapid and inexpensive method for implicating molecular targets.

Adams Dany S DS Levin Michael M

Genesis (New York, N.Y. : 2000) 20061101 11

Identification of gene products that function in some specific process of interest is a common goal in developmental biology. Although use of drug compounds to probe biological systems has a very long history in teratology and toxicology, systematic hierarchical drug screening has not been capitalized upon by the developmental biology community. This "chemical genetics" approach can greatly benefit the study of embryonic and regenerative systems, and we have formalized a strategy for using known ...[more]

PMID: 17078061

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Project description:BackgroundKoi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment.ResultsA rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers--two inner primers, two outer primers and two loop primers--was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65 degrees C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV.ConclusionThis paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath.

Dataset Information

Inverse drug screens: a rapid and inexpensive method for implicating molecular targets.

Publications

Inverse drug screens: a rapid and inexpensive method for implicating molecular targets.

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