Granted Patent B2

US 12,365,891 · App. 17/269,182 · Granted Jul 22, 2025

Inventors: Vera Baraznenok (Sunnyvale, CA); Alex I. Kutyavin (Sunnyvale, CA); Oliver Z. Nanassy (Sunnyvale, CA); Dmitri Sergueev (Sunnyvale, CA); Alexander A. Gall (Sunnyvale, CA)

Assignee: CEPHEID

C12N15/1006C12Q1/6806C08B37/0045

Abstract

Claims (60)

1. A method for isolation of a nucleic acid from a sample comprising a nucleic acid, comprising:

(a) contacting a sample comprising a nucleic acid with an aqueous composition comprising a polysaccharide comprising one or more repeating units having the structure of Formula II:

or a stereoisomer, a salt, a tautomer, or a combination thereof, wherein:

R 2 and R 3 are independently H, substituted C 1 -C 8 alkyl, substituted C 3 -C 8 cycloalkyl, substituted C 3 -C 8 heterocycloalkyl, or substituted C 2 -C 20 heteroalkyl; and

(b) concentrating the nucleic acid on a solid support thereby isolating the nucleic acid, wherein the method is performed in a cartridge.

2. The method of claim 1 , wherein the polysaccharide further comprises one or more repeating units of Formula (III):

or a stereoisomer, a salt, a tautomer, or a combination thereof, wherein R 3 is H, CH 3 , CH 2 CH 2 NH 2 , CH 2 CH 2 N(CH 3 ) 2 , CH 2 CH 2 OH, (CH 2 ) 2 O(CH 2 ) 2 NH 2 , or CH 2 CH 2 NHCH 2 CH 2 NH 2 .

3. The method of claim 1 , wherein the polysaccharide comprises one or more repeating units having the structure of Formula VI:

or a stereoisomer, a salt, a tautomer, or a combination thereof, wherein:

n is 0, 1, 2, or 3;

R 4 is H or C 1 -C 3 alkyl;

X, at each occurrence, is independently C 2 -C 4 alkylene or C 4 -C 6 heteroalkylene;

Y is a C 2 -C 3 alkylene or C 4 -C 6 heteroalkylene; and

R 5 and R 6 are independently H or C 1 -C 3 alkyl.

4. The method of claim 1 , wherein the polysaccharide comprises one or more repeating units having the structure of Formula V:

or a stereoisomer, a salt, a tautomer, or a combination thereof, wherein:

n is 0, 1, 2, or 3;

m, at each occurrence, is independently 2, 3, or 4;

p is 2, 3, or 4;

R 4 is H or C 1 -C 3 alkyl; and

R 5 and R 6 are independently H or C 1 -C 3 alkyl.

5. The method of claim 1 , wherein the polysaccharide comprises one or more repeating units represented by Formula VI, Formula VII, or Formula VIII:

or a stereoisomer, a salt, a tautomer, or combination thereof.

6. The method of claim 1 , wherein the polysaccharide is present in the aqueous composition at a concentration from about 0.1 μg/mL to about 1000 μg/mL.

7. The method of claim 1 , wherein the polysaccharide has a relative molecular weight between about 120 kDa and about 500 kDa.

8. The method of claim 1 , wherein the nucleic acid is concentrated by centrifugation, precipitation, or a combination thereof.

9. The method of claim 1 , wherein the nucleic acid is concentrated by precipitation on a solid support.

10. The method of claim 1 , wherein the solid support comprises a material selected from silica, glass, ethylenic backbone polymer, mica, polycarbonate, zeolite, titanium dioxide, or a combination thereof.

11. The method of claim 1 , wherein the solid support is a magnetic bead, glass bead, cellulose filter, polycarbonate filter, polytetrafluoroethylene filter, polyvinylpyrrolidone filter, polyethersulfone filter, or glass filter.

12. The method of claim 1 , wherein the method further comprises washing and/or eluting the nucleic acid concentrated on the solid support with a wash agent, an eluting agent, or a comniation thereof.

13. The method of claim 1 , wherein the aqueous composition further comprises one or more of a lysis agent, a chaotropic agent, a salt, a buffering agent, a surfactant, or a defoaming agent.

14. The method of claim 1 , wherein the sample comprising nucleic acid is or comprises blood, plasma, serum, semen, spinal fluid, tissue biopsy, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, PCR reaction mixture, cell lysate preparation, or in vitro nucleic acid modification reaction mixture.

15. A method for isolation of a nucleic acid from a sample comprising a nucleic acid, comprising:

(a) contacting a sample comprising a nucleic acid with an aqueous composition comprising a polysaccharide comprising one or more repeating units having the structure of Formula II:

or a stereoisomer, a salt, a tautomer, or a combination thereof, wherein:

R 2 and R 3 are independently H, substituted C 1 -C 8 alkyl, substituted C 3 -C 8 cycloalkyl, substituted C 3 -C 8 heterocycloalkyl, or substituted C 2 -C 20 heteroalkyl; and

(b) concentrating the nucleic acid on a solid support thereby isolating the nucleic acid, further comprising contacting the sample with a lysis buffer prior to contacting the sample with the aqueous composition.

16. The method of claim 15 , wherein the polysaccharide comprises one or more repeating units having the structure of Formula IV:

or a stereoisomer, a salt, a tautomer, or a combination thereof, wherein:

n is 0, 1, 2, or 3;

R 4 is H or C 1 -C 3 alkyl;

X, at each occurrence, is independently C 2 -C 4 alkylene or C 4 -C 6 heteroalkylene;

Y is a C 2 -C 3 alkylene or C 4 -C 6 heteroalkylene; and

R 5 and R 6 are independently H or C 1 -C 3 alkyl.

17. The method of claim 15 , wherein the polysaccharide comprises one or more repeating units represented by Formula VI, Formula VII, or Formula VIII:

or a stereoisomer, a salt, a tautomer, or combination thereof.

18. A method for detecting a nucleic acid in a sample, comprising:

(a) contacting the sample with an aqueous composition comprising a polysaccharide comprising one or more repeating units having the structure of Formula II:

or a stereoisomer, a salt, a tautomer, or a combination thereof, wherein:

R 2 and R 3 are independently H, substituted C 1 -C 8 alkyl, substituted C 3 -C 8 cycloalkyl, substituted C 3 -C 8 heterocycloalkyl, or substituted C 2 -C 20 heteroalkyl;

(b) concentrating the nucleic acid; and

(c) detecting the nucleic acid,

wherein detecting the nucleic acid comprises amplifying the nucleic acid by polymerase chain reaction.

19. The method of claim 18 , wherein the polysaccharide comprises one or more repeating units having the structure of Formula IV:

or a stereoisomer, a salt, a tautomer, or a combination thereof, wherein:

n is 0, 1, 2, or 3;

R 4 is H or C 1 -C 3 alkyl;

X, at each occurrence, is independently C 2 -C 4 alkylene or C 4 -C 6 heteroalkylene;

Y is a C 2 -C 3 alkylene or C 4 -C 6 heteroalkylene; and

R 5 and R 6 are independently H or C 1 -C 3 alkyl.

Assignments (1)

Continuity (2)

References Cited (72)