# US 8,784,627 B2 · Graft copolymers, their preparation and use in capillary electrophoresis

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- Number: US 8,784,627 B2
- Document ID: US8784627B2
- Status: Granted
- Kind: B2
- Application number: 12/565,590
- Filed: 2009-09-23
- Published: 2010-07-29
- Granted: 2014-07-22
- Earliest claimed: 2002-07-29
- Assignees: Applied Biosystems, LLC
- Applicants: Aldrich N. Lau
- Inventors: Aldrich N. Lau
- Web page: https://patentgrep.com/patent/US8784627B2

## CPC

- C08L 51/003: {grafted on to macromolecular compounds obtained by reactions only involving unsaturated carbon-to-carbon bonds (C08L51/04, C08L51/06 take precedence)}
- C08F 2/32: Polymerisation in water-in-oil emulsions
- C08F 120/54: Amides {, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide}
- C08F 120/56: Acrylamide; Methacrylamide
- C08F 220/54: Amides {, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide}
- C08F 265/10: on to polymers of amides or imides
- C08F 267/10: on to polymers of amides or imides
- C08L 33/24: Homopolymers or copolymers of amides or imides
- C08L 33/26: Homopolymers or copolymers of acrylamide or methacrylamide
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 27/44747: {Composition of gel or of carrier mixture}
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)
- C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention relates to graft copolymers, their preparation, and compositions, such as electrophoresis separation media, containing the same; also to ultra-high molecular weight poly(N,N-dimethylacrylamide) (“poly(DMA)”) polymers, their preparation, and compositions, such as electrophoresis separation media, containing the same; and more particularly to supports, such as capillaries, containing these polymers and methods for separating biomolecules, especially polynucleotides, using capillary electrophoresis. The graft copolymers can be prepared by, e.g., grafting polyacrylamide units onto a poly(DMA) backbone. Separation media comprising such graft copolymers or ultra-high molecular weight poly(DMA) polymers yield superior performance in the analysis and separation of biomolecules by capillary electrophoresis.

## Claims

### Claim 1

A method for separating a mixture of biomolecules, comprising:
(1) contacting a composition comprising a buffer and an effective amount of a poly(M1-g-M2), or a salt thereof, wherein:
(a) poly(M1-g-M2) comprises a first backbone polymer, poly (M1), formed from a monomer unit M1 having the formula (I):
wherein each A1 is independently O, S or NX1;
each of R1, R2, R3 and R4 is independently H, C1-C20 alkyl, C4-C12 cycloalkyl, C5-C12 aryl, C4-C12 heteroaryl, —(C1-C20 alkyl)(C5-C12 aryl) or —(C5-C12 aryl)(C1-C20 alkyl);
each R5 is independently C1-C20 alkyl, C1-C20 heteroalkyl, C4-C12 cycloalkyl, C4-C12 heterocycloalkyl, C5-C12 aryl, C4-C12 heteroaryl, —(C1-C20 alkyl)(C4-C12 cycloalkyl), —(C4-C12 cycloalkyl)(C1-C20 alkyl), —(C1-C20 heteroalkyl)(C4-C12 cycloalkyl), —(C4-C12 cycloalkyl)(C1-C20 heteroalkyl), —(C1-C20 alkyl)(C4-C12 heterocycloalkyl), —(C4-C12 heterocycloalkyl)(C1-C20 alkyl), —(C1-C20 heteroalkyl)(C4-C12 heterocycloalkyl), —(C4-C12 heterocycloalkyl)(C1-C20 heteroalkyl), —(C1-C20 alkyl)(C5-C12 aryl), —(C5-C12 aryl)(C1-C20 alkyl), —(C1-C20 heteroalkyl)(C5-C12 aryl), —(C5-C12 aryl)(C1-C20 heteroalkyl), —(C1-C20 alkyl)(C4-C12 heteroaryl), —(C4-C12 heteroaryl)(C1-C20 alkyl), —(C1-C20 heteroalkyl)(C4-C12 heteroaryl), —(C4-C12 heteroaryl)(C1-C20 heteroalkyl), —(C1-C4 alkyl)qNH2, —(C1-C4 alkyl)qCONH2, —(C1-C4 alkyl)NHCONH2, —(C1-C4 alkyl)NHCOH or —(C1-C4 alkyl)qNHCOCH3, where each q is 0 or 1; and
each X1 is independently H, C1-C20 alkyl, C4-C12 cycloalkyl, C5-C12 aryl, C4-C12 heteroaryl, —(C1-C20 alkyl)(C5-C12 aryl), —(C5-C12 aryl)(C1-C20 alkyl), —(C1-C4 alkyl)qNH2, —(C1-C4 alkyl)qCONH2, —(C1-C4 alkyl)NHCONH2, —(C1-C4 alkyl)qNHCOH or —(C1-C4 alkyl)qNHCOCH3, where each q is 0 or 1;
(b) poly(M1-g-M2) comprises a second pendent polymer, poly (M2), grafted to poly(M1), wherein the pendent polymer consists of a polymer formed from monomer units M2 having the formula (II):
wherein each A2 is independently O, S or NX2;
each of R6, R7, R8 and R9 is independently H, C1-C20 alkyl, C4-C12 cycloalkyl, C5-C12 aryl, C4-C12 heteroaryl, —(C1-C20 alkyl)(C5-C12 aryl) or —(C5-C12 aryl)(C1-C20 alkyl);
each R10 is independently H, C1-C20 alkyl, C1-C20 heteroalkyl, C4-C12 cycloalkyl, C4-C12 heterocycloalkyl, C5-C12 aryl, C4-C12 heteroaryl, —(C1-C20 alkyl)(C4-C12 cycloalkyl), —(C4-C12 cycloalkyl)(C1-C20 alkyl), —(C1-C20 heteroalkyl)(C4-C12 cycloalkyl), —(C4-C12 cycloalkyl)(C1-C20 heteroalkyl), —(C1-C20 alkyl)(C4-C12 heterocycloalkyl), —(C4-C12 heterocycloalkyl)(C1-C20 alkyl), —(C1-C20 heteroalkyl)(C4-C12 heterocycloalkyl), —(C4-C12 heterocycloalkyl)(C1-C20 heteroalkyl), —(C1-C20 alkyl)(C5-C12 aryl), —(C5-C12 aryl)(C1-C20 alkyl), —(C1-C20 heteroalkyl)(C5-C12 aryl), —(C5-C12 aryl)(C1-C20 heteroalkyl), —(C1-C20 alkyl)(C4-C12 heteroaryl), —(C4-C12 heteroaryl)(C1-C20 alkyl), —(C1-C20 heteroalkyl)(C4-C12 heteroaryl), —(C4-C12 heteroaryl)(C1-C20 heteroalkyl), —(C1-C4 alkyl)qNH2, —(C1-C4 alkyl)qCONH2, —(C1-C4 alkyl)NHCONH2, —(C1-C4 alkyl)NHCOH or —(C1-C4 alkyl)qNHCOCH3, where each q is 0 or 1; and
each X2 is independently H, C1-C20 alkyl, C4-C12 cycloalkyl, C5-C12 aryl, C4-C12 heteroaryl, —(C1-C20 alkyl)(C5-C12 aryl), —(C5-C12 aryl)(C1-C20 alkyl), —(C1-C4 alkyl)qNH2, —(C1-C4 alkyl)qCONH2, —(C1-C4 alkyl)NHCONH2, —(C1-C4 alkyl)qNHCOH or —(C1-C4 alkyl)qNHCOCH3, where each q is 0 or 1;
with a mixture comprising a biomolecule; and
(2) applying an electric field to the composition in an amount sufficient to facilitate the separation of the biomolecule from the mixture.

### Claim 2 (depends on 1)

The method of claim 1, wherein the separation is performed within a capillary tube and two or more biomolecules are polynucleotides.

### Claim 3 (depends on 2)

The method of claim 2, wherein the separation has a crossover of at least 400 base pairs.

### Claim 4 (depends on 3)

The method of claim 3, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 5 (depends on 2)

The method of claim 2, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 6 (depends on 1)

The method of claim 1, wherein the composition further comprises a sieve polymer.

### Claim 7 (depends on 6)

The method of claim 6, wherein the sieve polymer is poly(acrylamide).

### Claim 8 (depends on 7)

The method of claim 7, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 9 (depends on 6)

The method of claim 6, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 10 (depends on 1)

The method of claim 1, wherein the poly(M1-g-M2) or a salt thereof has a weight-average molecular weight of from about 150,000 Daltons to about 20 MDaltons.

### Claim 11 (depends on 10)

The method of claim 10, wherein the composition further comprises a sieve polymer or a salt thereof having a weight-average molecular weight of from about 100,000 Daltons to about 5 MDaltons.

### Claim 12 (depends on 11)

The method of claim 11, wherein the sieve polymer is substantially linear poly(acrylamide).

### Claim 13 (depends on 12)

The method of claim 12, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 14 (depends on 11)

The method of claim 11, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 15 (depends on 10)

The method of claim 10, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 16 (depends on 10)

The method of claim 10, wherein the sum of the weight of all M2 units present in the poly(M1-g-M2) or a salt thereof divided by the sum of the weight of all M1 units present in the poly(M1-g-M2) or a salt thereof is at least about 0.1.

### Claim 17 (depends on 1)

The method of claim 1, wherein the buffer is an aqueous buffer.

### Claim 18 (depends on 17)

The method of claim 17, wherein the composition has a pH of from about 5 to about 11.

### Claim 19 (depends on 18)

The method of claim 18, wherein the composition further comprises formamide, urea, pyrrolidone, N-methylpyrrolidone or a mixture thereof.

### Claim 20 (depends on 18)

The method of claim 18, wherein the composition further comprises urea.

### Claim 21 (depends on 18)

The method of claim 18, wherein the composition further comprises formamide.

### Claim 22 (depends on 18)

The method of claim 18, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 23 (depends on 17)

The method of claim 17, wherein the composition has a pH of from about 7 to about 10.

### Claim 24 (depends on 17)

The method of claim 17, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 25 (depends on 1)

The method of claim 1, wherein M1 is N,N-dimethylacrylamide and M2 is acrylamide.

### Claim 26 (depends on 1)

The method of claim 1, wherein the sum of the weight of all M2 units present in the poly(M1-g-M2) or a salt thereof divided by the sum of the weight of all M1 units present in the poly(M1-g-M2) or a salt thereof is at least about 0.1.

### Claim 27 (depends on 1)

The method of claim 1, wherein the second pendent polymer, poly(M2), is grafted to poly(M1) through R3, R5, or R4, if R4 is not H.

### Claim 28 (depends on 1)

The method of claim 1, wherein at least one M1 is different from at least one M2.

### Claim 29 (depends on 1)

The method of claim 1, wherein M1 is different from M2.