Method for Detecting or Quantifying a Truncating Mutation
AssigneesUniversite De Strasbourg, Centre National De La Recherche Scientifique
InventorsAndrew David Griffiths, Alex Garvin, Kerstin Godelinde Blank
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Abstract
The present invention discloses a new method for detecting or quantifying a truncating mutation of a target gene in a subject, said method relying on the in vitro compartmentalization of single genetic constructs in aqueous droplets of a water-in-oil emulsion.
Claims52
A method for detecting or quantifying a truncating mutation of a target gene in a subject, said method relying on the in vitro compartmentalization of single genetic constructs in aqueous droplets of a water-in-oil emulsion and comprising:
- a) providing a DNA sample from the subject;
- b) assembling genetic constructs, each construct comprising a test sequence of said target gene, obtained from said DNA sample, operably linked with a promoter and a ribosome binding site, a first reporter system used to control the presence of said test sequence in the construct and/or the presence of a construct comprising said test sequence in a droplet, and a second reporter system used to detect the presence of a truncating mutation of said target gene, said first and second reporter systems generating distinct signals;
- c) compartmentalizing each genetic construct in a droplet by forming a water-in-oil emulsion;
- d) transcription and translation of each genetic construct in each droplet
- e) monitoring emitted signals from said first and second reporter systems in each droplet to detect or quantify truncating mutations of said target gene.
A method for detecting or quantifying a truncating mutation of a target gene in a subject, said method relying on the in vitro compartmentalization of single genetic constructs in aqueous droplets of a water-in-oil emulsion and comprising:
- a) providing a DNA sample from the subject;
- b) compartmentalizing each DNA molecule, from said DNA sample, comprising a test sequence of said target gene in first droplets by forming a water-in-oil emulsion;
- c) assembling genetic constructs, each construct comprising said test sequence operably linked with a promoter and a ribosome binding site, a marker gene of a first reporter system used to control the presence of said test sequence in the construct and/or the presence of a construct comprising said test sequence in a droplet, and a marker gene of a second reporter system used to detect the presence of a truncating mutation of said target gene, said first and second reporter systems generating distinct signals;
- d) fusing first droplets containing genetic constructs of step c) with second droplets containing an in vitro transcription and translation system;
- e) transcription and translation of each genetic construct in each fusion droplet
- f) monitoring emitted signals from said first and second reporter systems in each fusion droplet to detect or quantify truncating mutations of said target gene.
A genetic construct comprising a test sequence operably linked with a promoter and a ribosome binding site, a first marker gene operably linked with another promoter, and a second marker gene which is operably linked to the test sequence in order to be expressed in a single mRNA, said marker gene being downstream to said test sequence.
A genetic construct comprising a test sequence operably linked with a promoter and a ribosome binding site, a first reporter system which is an affinity system comprising two members, a first member appended on the 5′ end of the coding strand of the test sequence and a second member which is bound to the first member and is able to generate a signal, and a marker gene which is operably linked to the test sequence in order to be expressed in a single mRNA, said marker gene being downstream to said test sequence
A droplet from a water-in-oil emulsion containing a genetic construct comprising at least two reporter systems generating distinct signals.
Dependencies are inferred from the claim text.