DEVICE AND METHOD FOR PRODUCING A REPLICATE OR DERIVATIVE FROM AN ARRAY OF MOLECULES, AND APPLICATIONS THEREOF

• US 20120015844A1
• Filed: 09/02/2011
• Published: 01/19/2012
• Est. Priority Date: 03/06/2009
• Status: Active Grant

• Current Assignee: Biocopy GmbH
• Original Assignee: Albert Ludwigs University of Freiburg, Hahn-Schickard-Gesellschaft für angewandte Forschung e.V.
• Inventors: ZENGERLE, Roland, VON STETTEN, Felix, ROTH, Guenter, HOFFMANN, Jochen
• Granted Patent: US 9,725,758 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2535/00 Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

A method of producing a replicate or derivative of an array of molecules, the array having a spatial arrangement of separate samples of molecules, includes creating, for each sample, at least one spatially limited effective area which is separate from the effective areas of the other samples, a surface, provided with a binding adapter or binding properties, of a carrier bordering on the effective areas. The molecules are amplified by means of amplifying agents in the effective areas for creating replicates or derivatives of the samples. The replicates or derivatives of the samples are bound to the carrier by means of the binding adapter or the binding properties, so that a spatial arrangement of the replicates or derivatives of the samples on the carrier corresponds to the spatial arrangement of the samples in the array. The carrier having the copies of the samples is removed from the array.

Claims

1. A method of producing a replicate or derivative of an array of molecules, the array comprising a spatial arrangement of separate samples of molecules, the method comprising:

creating, for each sample, at least one spatially limited effective area which is separate from the effective areas of the other samples, a surface, provided with a binding adapter or binding properties, of a carrier bordering on the effective areas;

amplifying the molecules by means of amplifying agents in the effective areas for creating replicates or derivatives of the samples;

binding the replicates or derivatives of the samples to the carrier by means of the binding adapter or the binding properties, so that a spatial arrangement of the copies or derivatives of the samples on the carrier corresponds to the spatial arrangement of the samples in the array; and

removing the carrier comprising the replicates or derivatives of the samples from the array.

2. The method as claimed in claim 1,

wherein producing a spatially limited effective area comprises producing a spatially limited amplifying agent area, or

wherein the spatially limited amplifying agent areas are defined, at least in part, by micro- or nanostructures within an array substrate of the array or within the carrier, or

wherein the micro- or nanostructures comprise an unordered matrix based, in particular, on a filter membrane, on a hydrogel or on an aerogel, or wherein the micro- or nanostructures are based on an ordered three-dimensional substrate.

3. The method as claimed in claim 1,

wherein producing at least one spatially limited amplifying agent area for each sample comprises providing the samples in mutually associated separately recesses within the array substrate, introducing the amplifying agent into the recesses, and closing off the recesses by the carrier, or

wherein the spatially limited amplifying agent areas are separated, at least in part, by phase boundaries between two liquids, a liquid and a gas, or a physical boundary, in particular a lipid membrane, or

wherein the spatially limited amplifying agent areas are separated, at least in part, by phase boundaries between two liquids, a liquid and a gas, or a physical boundary, in particular a lipid membrane, and wherein producing at least one spatially limited amplifying agent area for each sample comprises providing the samples in mutually separated droplets of liquid which comprise the amplifying agent and which are fixed, in the spatial arrangement, on an array substrate of the array, a thinner-bodied liquid being arranged between the droplets of liquid, and arranging the carrier in relation to the array substrate such that the surface, provided with the binding adapter, of the carrier borders on the droplets of liquid.

4. The method as claimed in claim 2, wherein producing at least one spatially limited amplifying agent area comprises providing the carrier comprising at least one recess which is associated with each sample and comprises the binding adapter arranged therein, introducing the amplifying agent into the recesses, and closing off the recesses by means of the array substrate, so that the samples are exposed to the amplifying agent area.

5. The method as claimed in claim 1, wherein producing a spatially limited amplifying agent area for each sample comprises providing the sample within a sequencer chip or a nanowell plate.

6. The method as claimed in claim 1,

wherein the process of binding the replicates or derivatives to the carrier is performed simultaneously with the amplification, or

wherein the spatial limitation of the effective area is that the binding adapters are present on the carrier, as complementary primers, in the form of a primer array that may comprise a regular or irregular distribution of spots, the spot size and spot density on the carrier being equal to or smaller than that on the array.

7. The method as claimed in claim 1, wherein the spatial limitation of the effective area is effected by applying an energy field.

8. The method as claimed in claim 1, wherein the samples are provided in the form of molecules bound to particles.

9. The method as claimed in claim 1,

wherein the array of molecules is a non-synthetic array of biomolecules, or

wherein the molecules are single- or double-stranded oligonucleotides, polynucleotides, DNA or synthetic molecules analogous to DNA (PNA), or.

wherein the array comprises a sequencing process for deriving the genome, a sequencing process for deriving the transcriptome, a process of sequencing RNA, mRNA, tRNA, siRNA, or a process of sequencing mutations and variations, or

wherein by means of amplifying and binding to the carrier, copies are created which correspond to a DNA, a modified DNA, expressions of a DNA, an RNA, proteins or peptides;

or

wherein the amplifying agent effects a DNA amplification, in particular a polymerase chain reaction, an isothermal amplification, or a NASBA reaction, and the binding adapter comprises a matching primer, or

wherein the method further comprises monitoring any changes in physical or chemical parameters within the effective areas

10. The method as claimed in claim 1, wherein the positions of the samples comprise further molecules or DNA sequences or cells located thereat which are part of the sample or are immobilized and which are needed for generating derivatives, in particular expression vector sequences such as ori, promoters, ribosome binding sites, start codon, endoprotease cleaving sites, fusion sequences, reporter genes, terminators, antibiotics resistance genes, in-vitro translation systems, or cells.

11. The method as claimed in claim 1,

wherein primary, secondary and/or tertiary derivatives are generated, from a primary array or a replicate of the primary array, in that DNA is transcribed into RNA, the RNA is translated into protein, or in that a binder is enriched while using a produced protein, a produced RNA or a produced DNA or the copy thereof from a liquid phase, or in that a binder interacts, or

wherein a derivative is generated on the solid phase of a target array and is present there in an immobilized manner.

14. A method of sequencing a liquid-particle array, a replicate being created from the samples comprised on the particles as claimed in claim 8, and the replicate being sequenced in a sequencing device.

12. A utilization of a replicate or derivative that was produced while using a method of producing a replicate or derivative of an array of molecules, the array comprising a spatial arrangement of separate samples of molecules, the method comprising:

creating, for each sample, at least one spatially limited effective area which is separate from the effective areas of the other samples, a surface, provided with a binding adapter or binding properties, of a carrier bordering on the effective areas;

amplifying the molecules by means of amplifying agents in the effective areas for creating replicates or derivatives of the samples;

binding the replicates or derivatives of the samples to the carrier by means of the binding adapter or the binding properties, so that a spatial arrangement of the copies or derivatives of the samples on the carrier corresponds to the spatial arrangement of the samples in the array; and

removing the carrier comprising the replicates or derivatives of the samples from the array,

either

for associating a reaction between a binder, in particular a protein, antibody or antigen, and an original molecule, its replicate or its derivative, with the DNA sequence of the original molecule, in particular for genotype-phenotype coupling, or

for associating a reaction wherein the original molecule, its copy or its derivative catalyzes the conversion of a substrate, with the DNA sequence of the original molecule, in particular for genotype-phenotype coupling, or

for identifying a DNA sequence, a RNA sequence, a protein or a catalytic, signaling (e.g. enhancing, allosteric, inhibiting . . . ) or enzymatic (e.g. lytic, phosphatase activity, kinase activity . . . ) function of a DNA, RNA or protein, or

for identifying a DNA sequence, a RNA sequence or a peptidic sequence and for producing, identification or preparation of a product, in particular antibody, antigen, vaccine or antibiotic, on the basis of the DNA, RNA or peptidic sequence, or

for detecting reactions between a sample, a replicate or derivative thereof with an interacting molecule or particle, said detection being performed by an optical, electrochemical or magnetic sensor, and the interacting molecule or particle carrying a corresponding marker, or said detection being performed, without any marker, via the change in the evanescent field or a modified resonance frequency, or by employing optical tweezers, or by coupling the reaction with a change in absorption, in particular precipitation or change of color, or with the emission of light, in particular chemiluminescence, or

for performing reactions on the replicate or derivative of the array, a chamber or fluidic structure comprising connecting terminals being applied over the surface of the replicate or derivative, or the replicate or derivative being introduced into a corresponding chamber, it being possible to incubate the chamber at a specific temperature, and to replace liquids comprised within the chamber, or

for simultaneously performing reactions and detections on the replicate or derivative.

13. The utilization as claimed in claim 12, wherein an identical sequencing device that is used for detecting the sequencing is also used for detecting the reactions or wherein the utilization is within a device that is also employed for sequencing the array.

15. A device for producing a replicate or derivative of an array of molecules, the array comprising a spatial arrangement of separate samples of molecules, the device comprising:

a creator for creating, for each sample, at least one spatially limited effective area which is separate from the effective areas of the other samples, a surface, provided with a binding adapter or binding properties, of a carrier bordering on the effective areas;

an amplifier for amplifying the molecules by means of amplifying agents in the effective areas for creating replicates or derivatives of the samples, and for binding the replicates or derivatives of the samples to the carrier by means of the binding adapter or the binding properties, so that a spatial arrangement of the copies or derivatives of the samples on the carrier corresponds to the spatial arrangement of the samples; and

a remover for removing the carrier comprising the copies of the samples from the array.