Device and method for producing a replicate or derivative from an array of molecules, and applications thereof

• US 9,725,758 B2
• Filed: 09/02/2011
• Issued: 08/08/2017
• Est. Priority Date: 03/06/2009
• Status: Active Grant

• Current Assignee: Biocopy GmbH
• Original Assignee: Albert Ludwigs University of Freiburg
• Inventors: Zengerle, Roland, Roth, Guenter, Von Stetten, Felix, Hoffmann, Jochen
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US13/224,645
• Publication Number:
  US 20120015844A1
• Time in Patent Office: 2,167 Days
• Field of Search: 506 7
• 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, the effective areas being spatially limited liquid amplifying agent areas, wherein a surface, provided with a binding adapter, of a carrier borders on the effective areas and the binding adapter is immobilized and arranged over an entire area of the carrier;

amplifying the molecules with amplifying agents in the effective areas for creating replicates or derivatives of every one of the samples;

binding the replicates or derivatives of the samples to the carrier with the binding adapter, 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;

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

wherein

the method of creating at least one spatially limited effective area further comprises producing a spatially limited amplifying agent area;

the process of binding the replicates or derivatives to the carrier is performed simultaneously with the amplifying of the molecules; and

the method is performed independently of whether any biochemical sequences or information of the array are known or predetermined.

2. The method as claimed in claim 1, 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, and

the micro- or nanostructures comprise an unordered matrix based, 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 a separated recess within the array substrate, introducing the amplifying agent into the recesses, and closing off the recesses by the carrier, or

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 lipid membrane, or

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 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 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 spatial limitation of the effective area is that the binding adapter is 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

the molecules are single- or double-stranded oligonucleotides, polynucleotides, DNA or synthetic molecules analogous to DNA, or

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

by 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

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

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 spatially limited effective areas comprise further molecules or DNA sequences or cells located therein which are part of the sample or are immobilized and which are needed for generating derivatives including expression vector sequences 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

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

12. The method as claimed in claim 1, the method further comprising:

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

(ii) 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, for genotype-phenotype coupling, or

(ii) identifying a DNA sequence, a RNA sequence, a protein or a catalytic function, a signaling function, or an enzymatic function of a DNA, RNA or protein, or

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

(v) 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, precipitation or change of color, or with the emission of light, chemiluminescence, or

(vi) 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

(vii) simultaneously performing reactions and detections on the replicate or derivative.

13. The method as claimed in claim 12, wherein a sequencing device that is used for sequencing the array of molecules is also used for each of steps (i)-(vii).