# US 6,586,211 B1 · Method for producing polymers

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- Number: US 6,586,211 B1
- Document ID: US6586211B1
- Status: Granted
- Kind: B1
- Application number: 09/869,332
- Filed: 2001-07-26
- Granted: 2003-07-01
- Earliest claimed: 1999-02-19
- Assignees: FeBit Ferrarius Biotechnology GmbH
- Inventors: Peer F. Stähler; Cord F. Stähler; Manfred Müller
- Web page: https://patentgrep.com/patent/US6586211B1

## CPC

- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- 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)}
- B01J 19/0093: {Microreactors, e.g. miniaturised or microfabricated reactors (laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027)}
- B01L 3/502707: {characterised by the manufacture of the container or its components}
- B01L 3/5085: {for multiple samples, e.g. microtitration plates}
- B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- G03F 7/70216: {Mask projection systems}
- G16B 50/00: ICT programming tools or database systems specially adapted for bioinformatics
- B01J 2219/00317: Microwell devices, i.e. having large numbers of wells
- B01J 2219/00432: Photolithographic masks
- B01J 2219/00436: Maskless processes
- B01J 2219/00439: using micromirror arrays
- B01J 2219/00441: using lasers
- B01J 2219/00448: using microlens arrays
- B01J 2219/00479: Means for mixing reactants or products in the reaction vessels
- B01J 2219/00497: Features relating to the solid phase supports
- B01J 2219/00511: Walls of reactor vessels
- B01J 2219/00529: DNA chips
- B01J 2219/00585: Parallel processes
- B01J 2219/0059: Sequential processes
- B01J 2219/00596: Solid-phase processes
- B01J 2219/00603: Making arrays on substantially continuous surfaces
- B01J 2219/00605: the compounds being directly bound or immobilised to solid supports
- B01J 2219/00608: DNA chips
- B01J 2219/00621: by physical means, e.g. trenches, raised areas
- B01J 2219/00626: Covalent
- B01J 2219/00648: by the use of solid beads
- B01J 2219/00659: Two-dimensional arrays
- B01J 2219/00675: In-situ synthesis on the substrate
- B01J 2219/00689: using computers
- B01J 2219/00702: Processes involving means for analysing and characterising the products
- B01J 2219/00704: integrated with the reactor apparatus
- B01J 2219/00711: Light-directed synthesis
- B01J 2219/00722: Nucleotides
- B01J 2219/00725: Peptides
- B01L 2300/0654: Lenses; Optical fibres
- B01L 2300/069: Absorbents; Gels to retain a fluid
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B 40/10: Libraries containing peptides or polypeptides, or derivatives thereof
- C40B 50/14: Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- G03F: PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR; (phototypographic composing devices B41B; photosensitive materials or processes for photographic purposes G03C; electrophotography, sensitive layers or processes therefor G03G)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

The invention relates to a method for producing polymers, in particular synthetic nucleic acid double strands of optional sequence, comprising the steps: (a) provision of a support having a surface area which contains a plurality of individual reaction areas, (b) location-resolved synthesis of nucleic acid fragments having in each case different base sequences in several of the individual reaction areas, and (c) detachment of the nucleic acid fragments from individual reaction areas.

## Claims

### Claim 1

Method for synthesizing polymers, wherein said method comprises synthesizing a plurality of oligomeric building blocks by parallel synthesis steps, wherein each oligomeric building block is synthesized on a different area of a common support, detaching the plurality of oligomeric building blocks from the support and bringing the oligomeric building blocks into contact with one another to synthesize the polymer, and wherein said different areas of said common support are at least partially in fluid communication during synthesis.

### Claim 2 (depends on 1)

Method according to claim 1, wherein double-stranded nucleic acid polymers of at least 300 bp in length are synthesized.

### Claim 3 (depends on 1)

A method according to claim 1, wherein the polymers are nucleic acid polymers which are selected from the group consisting of genes or sections thereof, gene clusters or sections thereof, chromosomes or sections thereof, and viral and bacterial genomes or sections thereof.

### Claim 4 (depends on 1)

Method according to claim 1, wherein the oligomeric building blocks are from 5 to 150 monomer units in length.

### Claim 5 (depends on 1)

Method according to claim 1, wherein partially complimentary oligonucleotide building blocks are detached from the support and are brought into contact with one another or with a polymer intermediate under hybridization conditions in successive steps.

### Claim 6 (depends on 1)

Method according to claim 1 for producing synthetic nucleic acid double strands, wherein said method comprises the steps:
(a) providing a support having a surface area which contains a plurality of individual reaction areas,
(b) synthesizing nucleic acid fragments in a location-resolved manner, wherein said nucleic acid fragments comprise different base sequences, as compared to other nucleic acid fragments, in several of the individual reaction areas, and
(c) detaching the nucleic acid fragments from the individual reaction areas.

### Claim 7 (depends on 6)

Method according to claim 6, wherein the base sequences of the nucleic acid fragments synthesized in individual reaction areas are chosen such that they can assemble to form a nucleic acid double strand hybrid.

### Claim 8 (depends on 6)

Method according to claim 6 wherein the nucleic acid fragments according to step (c) are detached in one or more steps under conditions such that a plurality of the detached nucleic acid fragments assemble to form a nucleic acid double strand hybrid.

### Claim 9 (depends on 8)

Method according to claim 8, wherein several nucleic acid fragments which form one strand of the nucleic acid double strand hybrid are linked covalently to one another.

### Claim 10 (depends on 9)

Method according to claim 9, wherein the covalent linking includes treatment with ligase or/and filling in gaps in the strands using DNA polymerase.

### Claim 11 (depends on 6)

Method according to claim 6, wherein the sequence comprises at one or more position recognition sequences for specific interaction with other molecules.

### Claim 12 (depends on 6)

Method according to claim 6, wherein the sequence of the nucleic acid double strands is a naturally occurring sequence, a not naturally occurring sequence or a combination of these two.

### Claim 13 (depends on 6)

Method according to claim 6, wherein the sequence is taken from a database, a sequencing experiment or a device for the integrated synthesis and analysis of polymers.

### Claim 14 (depends on 1)

Method according to claim 1, wherein the oligomeric building blocks are synthesized by location- or/and time-resolved illumination by means of a programmable light source matrix.

### Claim 15 (depends on 1)

Method according to claim 1, wherein the synthesizing step is a location- or/and time-resolved synthesis of the oligomeric building blocks in a microfluidic reaction support having one or more fluidic reaction compartments and one or more reaction areas within a fluidic reaction compartment.

### Claim 16 (depends on 1)

Method according to claim 1, wherein the oligomeric building blocks contain nucleotides occurring in nature, modified nucleotides or mixtures thereof.

### Claim 17 (depends on 1)

Method according to claim 1, wherein said oligomeric building blocks contain synthesis building blocks carrying labeling groups for subsequent detection of the polymer.

### Claim 18 (depends on 17)

Method according to claim 17, wherein the labeling groups are detectable in a light-dependent manner.

### Claim 19 (depends on 1)

The method of claim 1, wherein said method further comprises stabilizing, condensing and/or topologically manipulating a nucleic acid double strand or nucleic acid double strand precursor during or following the assembly of the nucleic acid double strand.

### Claim 20 (depends on 19)

A method according to claim 19, where the stabilization, condensation or/and topological manipulation is carried out by functional molecules selected from the group consisting of histones, topoisomerases, DNA-binding agents, DNA-binding proteins, DNA-stabilizing agents, DNA-stabilizing proteins, DNA-condensing agents and DNA-condensing proteins.

### Claim 21 (depends on 1)

Method according to claim 1, wherein double-stranded nucleic acid polymers of at least 1000 bp in length are synthesized.

### Claim 22 (depends on 1)

Method according to claim 1, wherein partially complimentary oligonucleotide building blocks are detached from the support and are brought into contact with (1) one another or with second partially complimentary oligonucleotide building blocks to form a polymer intermediate or (2) with a polymer intermediate under hybridization conditions, to form an advanced polymer intermediate, and thereafter third partially complimentary oligonucleotide building blocks are detached and brought into contact with (1) fourth partially complimentary oligonucleotide building blocks or with third partially complimentary oligonucleotide building blocks to form a polymer intermediate or (2) with a polymer intermediate under hybridization conditions, to form an advanced polymer intermediate, and these steps are repeated.

### Claim 23 (depends on 11)

Method according to claim 11, wherein the other molecules are selected from the group consisting of proteins, nucleic acids, peptides, pharmaceuticals, saccharides, lipids, hormones, and organic compounds.

### Claim 24 (depends on 20)

Method according to claim 20, wherein the other functional molecules are selected from the group consisting of histones or topoisomerases.

### Claim 25

Method for synthesizing polymers that are greater than 10,000 bp in length, wherein said method comprises synthesizing a plurality of oligomeric building blocks by parallel synthesis steps, wherein each oligomeric building block is synthesized on a different area of a common support, detaching the plurality of oligomeric building blocks from the support and bringing the oligomeric building blocks into contact with one another to synthesize the polymer, and wherein said different areas of said common support are at least partially in fluid communication during synthesis.

### Claim 26 (depends on 1)

Method according to claim 1, wherein the oligomeric building blocks are from 5 to 30 monomer units in length.