EP-4735623-A1 - METHODS OF FUNCTIONALIZING MAGNETIC PARTICLES AND METHODS OF GENERATING AMPLICONS USING MAGNETIC PARTICLES

EP4735623A1EP 4735623 A1EP4735623 A1EP 4735623A1EP-4735623-A1

Abstract

This application relates to functionalized magnetic particles and methods of using magnetic particles to generate amplicons. In some examples, a method of modifying a magnetic particle comprising a first functional group includes contacting the magnetic article with a first molecule comprising a polymer coupled to second and third functional groups, wherein the first functional group reacts with the second functional group to form a bond via which the polymer is coupled to the magnetic particle. The method may include contacting the magnetic particle with a second molecule, the second molecule comprising an oligonucleotide coupled to a fourth functional group, wherein the third functional group reacts with the fourth functional group to form a bond via which the ligonucleotide is coupled to the magnetic particle.

Inventors

GAL, Teodora
VON HATTEN, Xavier
ARTIOLI, GIANLUCA
TOVEY, Will
NGUYEN, NAM
LA ROSA, Angelo

Assignees

Illumina, Inc.

Dates

Publication Date: 20260506
Application Date: 20240625

Claims (1)

IP-2554-PCT 47CX-386113-WO WHAT IS CLAIMED IS: 1. A method of modifying a magnetic particle comprising a first functional group, the method comprising: contacting the magnetic particle with a first molecule comprising a polymer coupled to second and third functional groups, wherein the first functional group reacts with the second functional group to form a bond via which the polymer is coupled to the magnetic particle; and contacting the magnetic particle with a second molecule, the second molecule comprising an oligonucleotide coupled to a fourth functional group, wherein the third functional group reacts with the fourth functional group to form a bond via which the oligonucleotide is coupled to the magnetic particle. 2. The method of claim 1, wherein the oligonucleotide comprises an amplification primer. 3. The method of claim 1 or claim 2, wherein the second and third functional groups are of the same type as one another. 4. The method of any one of claims 1 to 3, wherein the second and third functional groups are of different types than one another. 5. The method of any one of claims 1 to 4, wherein the polymer comprises a hydrogel. 6. The method of claim 5, wherein the hydrogel comprises polyacrylamide. 7. The method of any one of claims 1 to 6, wherein the magnetic particle has a diameter of about 100 nm to about 300 nm. 8. A method of modifying a magnetic particle coupled to a polymer comprising a first functional group, the method comprising: contacting the magnetic particle with a molecule comprising an oligonucleotide coupled to a second functional group, wherein the second functional group reacts with the ^ IP-2554-PCT 47CX-386113-WO first functional group to form a bond via which the oligonucleotide is coupled to the magnetic particle. 9. A composition made by the method of any one of claims 1 to 8. 10. A composition, comprising: a magnetic particle; a polymer coupled to the magnetic particle via a reaction product of a first functional group coupled to the magnetic particle and a second functional group coupled to the polymer; and a plurality of oligonucleotides coupled to the polymer via reaction products of third functional groups coupled to the polymer and fourth functional groups coupled to the oligonucleotides. 11. A method of using the composition of claim 10, the method comprising: hybridizing a template polynucleotide to a first oligonucleotide of the plurality; and amplifying the hybridized template polynucleotide using additional oligonucleotides of the plurality to generate a plurality of amplicons coupled to the polymer via reaction products of the third and fourth functional groups. 12. The method of claim 11, further comprising, after the amplifying, disposing the magnetic particle within a flow cell. 13. The method of claim 12, wherein disposing the magnetic particle within the flow cell comprises electrostatically attracting the magnetic particle to a region of the flow cell. 14. The method of claim 11, further comprising, before the amplifying, disposing the magnetic particle within a flow cell. 15. A device comprising: a flow cell; and the composition of claim 10 located within the flow cell. ^ IP-2554-PCT 47CX-386113-WO 16. The device of claim 15, wherein the flow cell comprises a positively charged surface to which the composition is noncovalently bound through an electrostatic force. 17. A composition, comprising: a magnetic particle; and a molecule coupled to the magnetic particle, the molecule comprising: a polymer coupled to the magnetic particle via a reaction product of first and second functional groups; and a plurality of polynucleotide amplicons coupled to the polymer via reaction products of third and fourth functional groups. 18. A device comprising: a flow cell; and the composition of claim 17 located within the flow cell. 19. The device of claim 18, wherein the flow cell comprises a positively charged surface to which the composition is noncovalently bound through an electrostatic force. 20. A method of using the device of claim 19, the method comprising: sequencing the plurality of polynucleotide amplicons within the flow cell. 21. A method of generating an amplicon, the method comprising: coupling to a first particle a first double-stranded polynucleotide comprising first and second strands hybridized to one another, the first strand comprising a first amplification adaptor, the second strand comprising a second amplification adaptor that is complementary to the first amplification adapter; contacting the first particle with a second particle, the second particle comprising a first amplification primer that is complementary to the first amplification adapter; using a recombinase to hybridize the first amplification primer to the first amplification adaptor of the first double-stranded polynucleotide; and extending the first amplification primer to generate a first amplicon of the first strand of the first double-stranded polynucleotide. ^ IP-2554-PCT 47CX-386113-WO 22. The method of claim 21, wherein extending the first amplification primer releases the first strand from the first particle. 23. The method of claim 21 or claim 22, wherein the second particle further comprises a second amplification primer that is complementary to the second amplification adaptor, the method further comprising using the second amplification primer to generate a second amplicon of the first strand of the first double-stranded polynucleotide. 24. The method of claim 23, further comprising disposing the second particle within a flow cell. 25. The method of claim 24, wherein disposing the second particle within the flow cell comprises electrostatically attracting the second particle to a region of the flow cell. 26. The method of claim 24 or claim 25, wherein the second amplicon is generated before the second particle is disposed within the flow cell. 27. The method of claim 24 or claim 25, wherein the second amplicon is generated after the second particle is disposed within the flow cell. 28. The method of any one of claims 24 to 27, further comprising sequencing the first amplicon of the first strand of the first double-stranded polynucleotide within the flow cell. 29. The method of any one of claims 21 to 28, wherein the first particle comprises a carboxyl group to which the first double-stranded polynucleotide adsorbs. 30. The method of any one of claims 21 to 29, wherein the first particle is magnetic, and wherein the second particle is not magnetic. 31. The method of claim 30, further comprising using a magnet to separate the first particle from the second particle. 32. The method of any one of claims 21 to 31, wherein the first particle comprises a solid phase reversible immobilization (SPRI) bead. ^ IP-2554-PCT 47CX-386113-WO 33. The method of any one of claims 21 to 32, wherein the first particle is coupled to a second double-stranded polynucleotide comprising additional first and second strands hybridized to one another, the first strand comprising the first amplification adaptor, the additional second strand comprising the second amplification adaptor; contacting the first particle with a third particle comprising the first amplification primer; using the recombinase to hybridize the first amplification primer of the third particle to the first amplification adaptor of the second double-stranded polynucleotide; and extending the first amplification primer to generate a first amplicon of the first strand of the second double-stranded polynucleotide. 34. A composition, comprising: a first particle coupled to a first double-stranded polynucleotide comprising first and second strands hybridized to one another, the first strand comprising a first amplification adaptor, the second strand comprising a second amplification adaptor that is complementary to the first amplification adapter; a second particle in contact with the second particle and comprising a first amplification primer that is complementary to the first amplification adapter; and a recombinase to hybridize the first amplification primer to the first amplification adaptor of the first double-stranded polynucleotide. 35. A kit, comprising: a first particle coupled to a first double-stranded polynucleotide comprising first and second strands hybridized to one another, the first strand comprising a first amplification adaptor, the second strand comprising a second amplification adaptor that is complementary to the first amplification adapter; a second particle comprising a first amplification primer that is complementary to the first amplification adapter; and a recombinase to hybridize the first amplification primer to the first amplification adaptor of the first double-stranded polynucleotide. ^

Description

IP-2554-PCT 47CX-386113-WO METHODS OF FUNCTIONALIZING MAGNETIC PARTICLES AND METHODS OF GENERATING AMPLICONS USING MAGNETIC PARTICLES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/511,129, filed on June 29, 2023 and entitled “Methods of Functionalizing Magnetic Particles and Methods of Generating Amplicons Using Magnetic Particles,” the entire contents of which are incorporated by reference herein. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] The material in the accompanying sequence listing is hereby incorporated by reference into the application. The accompanying sequence listing XML file, named “IP- 2554-PCT.xml”, was created on June 2, 2024 and is 12 kB in size. FIELD [0003] This application relates to methods of functionalizing particles and generating amplicons using particles. BACKGROUND [0004] Cluster amplification is an approach to amplifying polynucleotides, for example for use in genetic sequencing. Target polynucleotides are captured by primers (e.g., P5 and P7 primers) coupled to a substrate surface in a flowcell, and form “seeds” at random locations on the surface. Cycles of amplification are performed to form clusters on the surface around each seed. The clusters include copies, and complementary copies, of the seed polynucleotides. In some circumstances, the substrate is patterned so as to define regions that bound different clusters, such as wells that may be filled with respective clusters. SUMMARY [0005] Examples provided herein are related to functionalizing magnetic particles, and generating amplicons using magnetic particles. ^ IP-2554-PCT 47CX-386113-WO [0006] Some examples herein provide a method of modifying a magnetic particle including a first functional group. The method may include contacting the magnetic particle with a first molecule including a polymer coupled to second and third functional groups. The first functional group reacts with the second functional group to form a bond via which the polymer is coupled to the magnetic particle. The method may include contacting the magnetic particle with a second molecule. The second molecule may include an oligonucleotide coupled to a fourth functional group. The third functional group reacts with the fourth functional group to form a bond via which the oligonucleotide is coupled to the magnetic particle. [0007] In some examples, the oligonucleotide includes an amplification primer. [0008] In some examples, the second and third functional groups are of the same type as one another. In some examples, the second and third functional groups are of different types than one another. [0009] In some examples, the polymer includes a hydrogel. In some examples, the hydrogel includes polyacrylamide. [0010] In some examples, the magnetic particle has a diameter of about 100 nm to about 300 nm. [0011] Some examples herein provide a method of modifying a magnetic particle coupled to a polymer including a first functional group. The method includes contacting the magnetic particle with a molecule including an oligonucleotide coupled to a second functional group. The second functional group reacts with the first functional group to form a bond via which the oligonucleotide is coupled to the magnetic particle. [0012] Some examples herein provide a composition made by any of the foregoing methods. [0013] Some examples herein provide a composition that includes a magnetic particle, and a polymer coupled to the magnetic particle via a reaction product of a first functional group coupled to the magnetic particle and a second functional group coupled to the polymer. The composition also may include a plurality of oligonucleotides coupled to the polymer via reaction products of third functional groups coupled to the polymer and fourth functional groups coupled to the oligonucleotides. ^ IP-2554-PCT 47CX-386113-WO [0014] Some examples herein provide a method of using such a composition. The method may include hybridizing a template polynucleotide to a first oligonucleotide of the plurality. The method may include amplifying the hybridized template polynucleotide using additional oligonucleotides of the plurality to generate a plurality of amplicons coupled to the polymer via reaction products of the third and fourth functional groups. [0015] In some examples, the method may include, after the amplifying, disposing the magnetic particle within a flow cell. In some examples, disposing the magnetic particle within the flow cell includes electrostatically attracting the magnetic particle to a region of the flow cell. [0016] In some examples, the method may include, before the amplifying, disposing the magnetic particle within a flow cell. [0017] Some examples herein provide a device that include a flow cell; and any of the foregoing compositions located within the flow cell. [0018] In some examples, the flow cell includes a positively charged surface to which the composi