Back to results
US 10,683,389 B2Granted
Beads for nucleic acid sequencing
AssigneeGenapsys, Inc.
InventorsAldrich N. K. Lau, Paul M. Kenney, Henrik Persson
Life of this patent
Estimating the term…
Abstract
Provided herein are methods that comprise providing polymer chains comprising a plurality of reactive groups onto the surface of beads and covalent attachment of functionalized biomolecules, such as primers.
Claims26
A method for preparation of a primer-conjugated bead, comprising:
- (a) coupling a plurality of polymer chains comprising a plurality of first reactive groups to an aminated bead to provide a plurality of second reactive groups coupled to the bead through a respective one of the polymer chains;
- and
- (b) covalently attaching amine- or hydroxy-terminated primers to the plurality of second reactive groups to form the primer-conjugated bead, wherein an individual polymer chain of the plurality of polymer chains comprises a repeat unit comprising a functional group that is different from functional groups of the plurality of first reactive groups and the plurality of second reactive groups, wherein the functional group is hydrophilic, and wherein the individual polymer chain comprises greater than or equal to 40 mole % (mol %) of the functional group,
- wherein a given polymer chain of the plurality of polymer chains comprises a carbon backbone of the formula:
- wherein ‘T’ is selected from the group consisting of NH2, NH(CH3), N(CH3)2, NH(CH2)tOH, (OCH2CH2)vCH3, (OCH2CH2)vOH, (OCH2CH2)vOCH3 and OCH3, wherein ‘t’ is 1 to 4, ‘v’ is 1 to 100;
- wherein ‘R2’, ‘R3’, ‘R4’ and ‘R5’ are independently H or CH3;
- and
- wherein ‘y’ and ‘z’ are mole percentages, wherein (‘y’+‘z’)=100 mol %, and wherein ‘y’ is less than or equal to 60 mol % and ‘z’ is greater than or equal to 40 mol %.
5 more elements
A method for preparation of a primer-conjugated bead, comprising:
- (a) coupling a plurality of polymer chains comprising a plurality of first reactive groups to an aminated bead to provide a plurality of second reactive groups coupled to the bead through a respective one of the polymer chains, and
- (b) covalently attaching amine- or hydroxy-terminated primers to the plurality of second reactive groups to form the primer-conjugated bead, wherein an individual polymer chain of the plurality of polymer chains comprises a repeat unit comprising a functional group that is different from functional groups of the plurality of first reactive groups and the plurality of second reactive groups, wherein the functional group is hydrophilic, and wherein the individual polymer chain comprises greater than or equal to 40 mole % (mol %) of the functional group,
- wherein a given polymer chain of the plurality of polymer chains comprises a carbon backbone of the formula:
- wherein ‘X’ is a halide;
- wherein ‘R1’ is OH;
- (OCH2CH2)vOH with v=1 to 200;
- NH(CH2)aOH, with ‘a’=1 to 6;
- NH(CH2CH2O)tCH2CH2OH with ‘t’=0 to 200;
- or Fz, with ‘z’=1, 2, 3, 4 or 5;
- wherein ‘T’ is selected from the group consisting of NH2, NH(CH3), N(CH3)2, N(CH2CH3)2, NHCH2CH2(OCH2CH2)bOCH3 and (OCH2CH2)dOCH3, wherein ‘b’=0 to 200, ‘d’=1 to 200;
- wherein ‘R2’ and ‘R3’ are independently H or CH3;
- wherein ‘x’ and ‘y’ are mole percentages, wherein (‘x’+‘y’)=100 mol % and ‘x’ ranges from 0.01 to 100 mol %, and
- wherein ‘W’ is a single bond or
- wherein ‘Di’=H or CH3, and
- wherein ‘i’ is 1, 2, 3 or 4, independently.
12 more elements
A method for preparation of a primer-conjugated bead, comprising:
- (a) coupling a plurality of polymer chains comprising a plurality of first reactive groups to an aminated bead to provide a plurality of second reactive groups coupled to the bead through a respective one of the polymer chains;
- and
- (b) covalently attaching amine- or hydroxy-terminated primers to the plurality of second reactive groups to form the primer-conjugated bead, wherein an individual polymer chain of the plurality of polymer chains comprises a repeat unit comprising a functional group that is different from functional groups of the plurality of first reactive groups and the plurality of second reactive groups, wherein the functional group is hydrophilic, and wherein the individual polymer chain comprises greater than or equal to 40 mole % (mol %) of the functional group,
- wherein a given polymer chain of the plurality of polymer chains comprises a carbon backbone of the formula:
- wherein ‘X’ is a halide;
- wherein ‘R1’ is OH;
- (OCH2CH2)vOH with v=1 to 200;
- NH(CH2)aOH, with ‘a’=1 to 6;
- NH(CH2CH2O)tCH2CH2OH with ‘t’=0 to 200,
- with ‘Q’=NO2 or Fz, wherein ‘z’ is 1, 2, 3, 4 or 5;
- wherein ‘T’ is selected from the group consisting of NH2, NH(CH3), N(CH3)2, N(CH2CH3)2, NHCH2CH2(OCH2CH2)bOCH3 and (OCH2CH2)dOCH3, wherein ‘b’=0 to 200, ‘d’=1 to 200;
- wherein ‘R2’ and ‘R3’ are independently H or CH3;
- wherein ‘y’, ‘p’, ‘q’ and ‘x’ are mole percentages, wherein (‘x’+‘y’)=100 mol %, (‘p’+‘q’)=x, x ranges from 0.01 to 100 mol % and ‘p’ ranges from 0.01 to 100 mol %, and
- wherein ‘W’ is a single bond or
- wherein ‘Di’ is H or CH3, and wherein ‘i’ is 1, 2, 3 or 4, independently.
12 more elements
A method for preparation of a primer-conjugated bead, comprising:
- (a) coupling a plurality of polymer chains comprising a plurality of first reactive groups to an aminated bead to provide a plurality of second reactive groups coupled to the bead through a respective one of the polymer chains;
- and
- (b) covalently attaching amine- or hydroxy-terminated primers to the plurality of second reactive groups to form the primer-conjugated bead, wherein an individual polymer chain of the plurality of polymer chains comprises a repeat unit comprising a functional group that is different from functional groups of the plurality of first reactive groups and the plurality of second reactive groups, wherein the functional group is hydrophilic, and wherein the individual polymer chain comprises greater than or equal to 40 mole % (mol %) of the functional group,
- wherein a given polymer chain of the plurality of polymer chains comprises a carbon backbone of the formula:
- wherein ‘X’ is a halide;
- wherein ‘R2’, ‘R3’, ‘R4’ and ‘R5’ are independently H or CH3;
- wherein ‘T’ is selected from the group consisting of NH2, NH(CH3), N(CH3)2, N(CH2CH3)2, NHCH2CH2(OCH2CH2)bOCH3 and (OCH2CH2)dOCH3, wherein ‘b’=0 to 200, ‘d’=1 to 200;
- and
- wherein ‘x’ and ‘y’ are mole percentages, wherein (‘x’+‘y’)=100 mol % and ‘x’ ranges from 0.01 to 100 mol %.
6 more elements
A method for preparation of a primer-conjugated bead, comprising:
- (a) coupling a plurality of polymer chains comprising a plurality of first reactive groups to an aminated bead to provide a plurality of second reactive groups coupled to the bead through a respective one of the polymer chains;
- and
- (b) covalently attaching amine- or hydroxy-terminated primers to the plurality of second reactive groups to form the primer-conjugated bead, wherein an individual polymer chain of the plurality of polymer chains comprises a repeat unit comprising a functional group that is different from functional groups of the plurality of first reactive groups and the plurality of second reactive groups, wherein the functional group is hydrophilic, and wherein the individual polymer chain comprises greater than or equal to 40 mole % (mol %) of the functional group,
- wherein a given polymer chain of the plurality of polymer chains comprises a carbon backbone of the formula:
- wherein ‘X’ is a halide;
- wherein ‘R2’, ‘R3’, ‘R4’ and ‘R5’ are independently H or CH3;
- wherein ‘T’ is selected from the group consisting of NH2, NH(CH3), N(CH3)2, N(CH2CH3)2, NHCH2CH2(OCH2CH2)bOCH3 and (OCH2CH2)dOCH3, wherein ‘b’=0 to 200, ‘d’=1 to 200;
- and
- wherein ‘y’, ‘p’, ‘q’ and ‘x’ are mole percentages, wherein (‘x’+‘y’)=100 mol %, (‘p’+‘q’)=‘x’, ‘x’ ranges from 0.01 to 100 mol % and ‘p’ ranges from 0.01 to 100 mol %.
6 more elements
A method for coupling primers to a bead, comprising:
- (a) providing a bead comprising amine groups linked to a surface of the bead;
- (b) reacting at least a portion of the amine groups with polymer chains comprising a carbon backbone of the formula:
- wherein ‘T’ is selected from the group consisting of NH2, NH(CH3), N(CH3)2, NH(CH2)tOH, (OCH2CH2)vCH3, (OCH2CH2)vOH, (OCH2CH2)vOCH3 and OCH3, wherein ‘t’ is 1 to 4, ‘v’ is 1 to 100;
- wherein ‘R2’, ‘R3’, ‘R4’ and ‘R5’ are independently H or CH3;
- and
- wherein ‘y’ and ‘z’ are mole percentages, wherein (‘y’+‘z’)=100 mol %, wherein ‘y’ is less than or equal to 60 mol %, ‘z’ is greater than or equal to 40 mol %;
- and
- (c) reacting the immobilized polymer chains with a primer comprising an amine group, providing immobilized primers coupled to at least a portion of the immobilized polymer chains.
5 more elements
Dependencies are inferred from the claim text.