US 9,068,972 B2 · GFP mutagenesis amplification: use of a fluorescence-antibiotic resistance fusion dual reporter construct to provide quantitative and highly sensitive detection of mutations

Abstract

A reversion mutation assay that is unique in providing a quantitative readout for mutagenesis. This assay is based on the creation of a functional GFP-β-lactamase fusion protein as a reporter providing both antibiotic resistance and fluorescence. This dual reporter is placed in a multicopy plasmid to increase the number of targets, with a reversion site at the N-terminus. Rare mutations at the reversion site allow read-through of the fusion protein, producing both beta-lactamase (providing antibiotic resistance) and GFP (emitting fluorescence). In the presence of carbenicillin, beta-lactamase production confers a selective advantage that allows amplification of mutant plasmids, raising the level of fluorescence emitted by GFP to levels that are detectable by fluorimetry. A window of time can be found where fluorescence is proportional to the number of mutation events at the reversion site, making fluorescence a quantitative measure of mutagenesis. Quantitative (as opposed to binary) detection of mutations allows substantial savings in test sample. This has applications in drug discovery, allowing high-throughput screening for DNA-targeting compounds and early pre-screening of leads for potential carcinogenic activity. The increased sensitivity of this assay also facilitates monitoring complex environmental samples.

Claims19

Dependent claims

A method for detecting and quantitatively measuring the mutagenic potency of a test substance, the method comprising:

a) providing a test substance,
b) providing a functional quantitative reporter-antibiotic resistance fusion construct to act as a dual reporter, wherein the dual reporter comprises an antibiotic reversion reporter and a quantitative reporter fused together in the same open reading frame and under the control of the same promoter, wherein the dual reporter possesses a reversion site at the N-terminus of the quantitative reporter-antibiotic resistance operon, wherein the dual reporter is cloned into a multicopy plasmid, and wherein mutations at the reversion site allow read-through of the fusion protein producing both an antibiotic resistance protein and a quantitative reporter protein, and further wherein, in the presence of an antibiotic, the production of the antibiotic resistance protein confers a selective advantage that causes amplification of mutant plasmids, thereby raising the level of quantitative reporter protein wherein the level of quantitative reporter protein is proportional to the number of mutation events at the reversion site,
c) transforming a host cell with the multicopy plasmid,
d) contacting the host cell to the test substance to induce mutations at the reversion site,
e) amplifying the plasmids that have mutations at the reversion site,
f) measuring the level of quantitative reporter protein, wherein the level of quantitative reporter protein is quantitatively proportional to the number of mutation events at the reversion site and therefore to the mutagenic potency of the test substance.

3 more elements

Dependencies are inferred from the claim text.

GFP mutagenesis amplification: use of a fluorescence-antibiotic resistance fusion dual reporter construct to provide quantitative and highly sensitive detection of mutations

Life of this patent

GFP mutagenesis amplification: use of a fluorescence-antibiotic resistance fusion dual reporter construct to provide quantitative and highly sensitive detection of mutations

Life of this patent

Abstract

Claims19