Methods and compositions for single molecule composition loading

• US 10,300,452 B2
• Filed: 03/23/2016
• Issued: 05/28/2019
• Est. Priority Date: 03/24/2015
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Sun, Lei, Popovich, Natasha, Shen, Gene, Pham, Thang, Turner, Stephen
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US15/078,915
• Publication Number:
  US 20160310926A1
• Time in Patent Office: 1,161 Days
• CPC Class Codes: 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
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/0074 Biological products
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/203 incorporating a composite nucleic acid containing a polypeptide sequence other than PNA
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2563/131 : the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 : being a biochannel or pore

Abstract

The present invention provides methods, compositions, and systems for distributing single polymerase molecules into array regions. In particular, the methods, compositions, and systems of the present invention result in a distribution of single polymerase molecules into array regions at a percentage that is larger than the percentage expected to be occupied under a Poisson distribution.

Claims

1. A method of distributing single polymerase molecules into array regions, the method comprising;

(a) providing a surface comprising a plurality of array regions, wherein each array region comprises several binding elements;

(b) exposing the surface to a solution comprising polymerase enzyme compositions, wherein each polymerase enzyme composition comprises a polymerase bound to a scaffold, wherein the scaffold comprises;

(i) a core comprising a material other than DNA, and

(ii) at least three linear arms comprising biotin moieties attached to DNA molecules, wherein each arm comprises multiple biotin moieties,

wherein the exposing is conducted under conditions such that the biotin moieties of the scaffold react with available binding elements in a given array region and prevent other polymerase enzyme compositions from loading in that given array region,

thereby distributing single polymerase molecules into array regions.

2. The method of claim 1, wherein the biotin moieties are attached to DNA molecules through attachment to a nucleobase.

5. The method of claim 1, wherein the multiple arms have a length of from about 0.5 nm to about 3 nm.

6. The method of claim 1, wherein the multiple arms have a length of about 1 nm.

7. The method of claim 1, wherein the multiple arms have a length of about 2 nm.

10. The method of claim 1, wherein each arm comprises 5-40 biotin moieties.

12. The method of claim 1, wherein the biotin moieties are attached to the DNA molecules by a method selected from the group consisting of:

hybridization, NHS ester chemistry, maleimide chemistry, and click chemistry.

13. The method of claim 1, wherein the distributing of the single polymerase molecules results in more than 37% of the array regions containing a single polymerase molecule.

14. The method of claim 1, wherein the scaffold comprises at least 4 arms.

15. The method of claim 1, wherein prior to the exposing step (b), the surface is functionalized with avidin moieties.

16. The method of claim 1, wherein the surface comprises biotin moieties overlaid with an avidin coating.

17. The method of claim 1, wherein the array regions comprise zero mode waveguides (ZMWs).

3. The method of 1, wherein the biotin moieties are attached to DNA molecules via a modified base.

4. The method of claim 3, wherein the modified base is a member selected from the group consisting of aminoallyl-dT, aminopropargyl-dT, thiol-phosphate, thiol-modified base, azide modified base, and an alkyl-modified base.

8. A method of distributing single polymerase molecules into array regions, the method comprising;

(a) providing a surface comprising a plurality of array regions, wherein each array region comprises several binding elements;

(b) exposing the surface to a solution comprising polymerase enzyme compositions, wherein each polymerase enzyme composition comprises a polymerase bound to a scaffold, wherein the scaffold comprises;

(i) a core, wherein the core comprises polyethylene glycol, and

(ii) multiple arms comprising biotin moieties attached to DNA molecules,

wherein the exposing is conducted under conditions such that the biotin moieties of the scaffold react with available binding elements in a given array region and prevent other polymerase enzyme compositions from loading in that given array region,

thereby distributing single polymerase molecules into array regions.

9. A method of distributing single polymerase molecules into array regions, the method comprising;

(a) providing a surface comprising a plurality of array regions, wherein each array region comprises several binding elements;

(b) exposing the surface to a solution comprising polymerase enzyme compositions, wherein each polymerase enzyme composition comprises a polymerase bound to a scaffold, wherein the scaffold comprises;

(i) a core, wherein the core comprises a multi-armed polyethylene glycol molecule, and

(ii) multiple arms comprising biotin moieties attached to DNA molecules,

wherein the exposing is conducted under conditions such that the biotin moieties of the scaffold react with available binding elements in a given array region and prevent other polymerase enzyme compositions from loading in that given array region,

thereby distributing single polymerase molecules into array regions.

11. A method of distributing single polymerase molecules into array regions, the method comprising;

(a) providing a surface comprising a plurality of array regions, wherein each array region comprises several binding elements;

(b) exposing the surface to a solution comprising polymerase enzyme compositions, wherein each polymerase enzyme composition comprises a polymerase bound to a scaffold, wherein the scaffold comprises;

(i) a core, and

(ii) multiple arms comprising biotin moieties attached to DNA molecules, wherein the biotin moieties are part of an oligonucleotide, and wherein the biotin moieties are attached to the DNA molecules through hybridization between the oligonucleotide and the DNA molecule,

wherein the exposing is conducted under conditions such that the biotin moieties of the scaffold react with available binding elements in a given array region and prevent other polymerase enzyme compositions from loading in that given array region,

thereby distributing single polymerase molecules into array regions.