# US 11,999,994 B2 · Methods for production and quantification of unique molecular identifier-labeled beads

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- Number: US 11,999,994 B2
- Document ID: US11999994B2
- Status: Granted
- Kind: B2
- Application number: 17/574,842
- Filed: 2022-01-13
- Published: 2022-07-14
- Granted: 2024-06-04
- Earliest claimed: 2021-01-14
- Assignees: Integrated Dna Technologies, Inc.
- Applicants: Integrated Dna Technologies, Inc.
- Inventors: Joseph Dobosy; Scott D. Rose; Jeffrey A. Manthey; Shawn D. Allen; Steven A. Henck; Mark Behlke
- Web page: https://patentgrep.com/patent/US11999994B2

## CPC

- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12N 2310/11: Antisense
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2320/11: for the determination of target sites, i.e. of active nucleic acids
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Described herein are compositions and methods for the production and quantification of barcoded or unique molecular identifier (UMI)-labeled substrates. In one aspect, the substrate is a bead comprising a template oligonucleotide that is elongated by successive extension reactions to provide a bead with an oligonucleotide comprising a plurality of barcodes and conserved anchor regions. Methods are also described for quantifying the amount of template oligonucleotide loaded onto the substrate and the products of the extension reaction after each round and after the final extension.

## Claims

### Claim 1

A method for preparing a unique molecular labeled substrate, the method comprising:
(a) providing a substrate comprising one or more oligonucleotide templates attached to the substrate,
wherein the substrate is a bead, derivatized bead, derivatized glass slide, or derivatized polymer;
(b) adding a primer template partially complementary to the one or more oligonucleotide templates;
wherein:
the primer template comprises a single stranded oligonucleotide having a structure comprising:
5′-Antisense-N8-20-Barcode-N8-20-Anchor-N4-20-x-3′,
where:
Antisense-N8-20 is an antisense sequence of 8 to 20 nucleotides complementary to an anchor sequence of the oligonucleotide template or the extended product:
Barcode-N8-20 is a barcode region of 8 to 20 nucleotides;
Anchor-N4-20 is an anchor sequence of 4 to 20 nucleotides; and
-x is a 3′-blocking moiety to prevent extension from the 3′-terminus of the primer template;
(c) adding reagents sufficient to perform an extension reaction;
(d) incubating the extension reaction for a period of time sufficient to produce an extension product;
(e) purifying the substrate comprising the extension product;
(f) repeating steps (b) to (e) at least two additional times using additional primer templates partially complementary to the extended product from each subsequent round;
(g) purifying the substrate comprising the final extension product using the same process as in step (e); and
(h) optionally, quantitating the amount of the oligonucleotide templates attached to the substrate, the amount of extension product attached to the substrate at each round, or the amount of the final extension product attached to the substrate.

### Claim 2 (depends on 1)

The method of claim 1, wherein a plurality of reactions is performed simultaneously using different primer templates partially complementary to the one or more oligonucleotide templates in step (b) and the additional primer templates partially complementary to the extended product from each subsequent round in step (f).

### Claim 3 (depends on 2)

The method of claim 2, wherein at least 2, 8, 12, 16, 24, 48, 96, 192, 384, 768, 1536, or more reactions are performed simultaneously.

### Claim 4 (depends on 1)

The method of claim 1, wherein the one or more oligonucleotide templates in step (a) comprises a conserved anchor sequence.

### Claim 5 (depends on 1)

The method of claim 1, wherein the one or more oligonucleotide templates in step (a) comprises at least one deoxyuridine nucleotide.

### Claim 6 (depends on 1)

The method of claim 1, wherein the one or more oligonucleotide templates in step (a) comprise a single stranded oligonucleotide have a structure comprising:
Substrate-Template-N20-50-Anchor-N4-20,
wherein:
Substrate is an inert substrate;
Template-N20-50 is a template sequence of 20 to 50 nucleotides optionally comprising at least one deoxyuridine nucleotide; and
Anchor-N4-20 is an anchor sequence of 4 to 20 nucleotides.

### Claim 7 (depends on 1)

The method of claim 1, wherein the one or more oligonucleotide templates in step (a) comprise a single stranded oligonucleotide have a structure comprising:
Substrate-Template-N20-50-Anchor-N8-20,
wherein:
Substrate is an inert substrate;
Template-N20-50 is a template sequence of 20 to 50 nucleotides optionally comprising at least one deoxyuridine nucleotide; and
Anchor-N8-20 is an anchor sequence of 8 to 20 nucleotides.

### Claim 8 (depends on 1)

The method of claim 1, wherein the primer template partially complementary to the one or more oligonucleotide templates in step (b) and the additional primer templates partially complementary to the extended product from each subsequent round in step (f) each comprise one or more unique barcodes and one or more conserved anchor sequences.

### Claim 9 (depends on 1)

The method of claim 1, wherein the additional primer templates partially complementary to the extended product from each subsequent round in step (f) comprise a single stranded oligonucleotide having a structure comprising:
5′-Antisense-N8-20-Barcode-N8-20-Anchor-N4-20-X-3′,
wherein:
Antisense-N8-20 is an antisense sequence of 8 to 20 nucleotides complementary to an anchor sequence of the oligonucleotide template or the extended product;
Barcode-N8-20 is a barcode region of 8 to 20 nucleotides;
Anchor-N4-20 is an anchor sequence of 4 to 20 nucleotides; and
-x is a 3′-blocking moiety to prevent extension from the 3′-terminus of the primer template.

### Claim 10 (depends on 1)

The method of claim 1, wherein the primer template partially complementary to the one or more oligonucleotide templates in step (b) and the additional primer templates partially complementary to the extended product from each subsequent round in step (f) comprise a single stranded oligonucleotide having a structure comprising:
5′-Antisense-N8-20-Barcode-N8-20-Anchor-N8-20-x-3′,
wherein:
Antisense-N8-20 is an antisense sequence of 8 to 20 nucleotides complementary to an anchor sequence of the oligonucleotide template or the extended product;
Barcode-N8-20 is a barcode region of 8 to 20 nucleotides;
Anchor-N8-20 is an anchor sequence of 8 to 20 nucleotides; and
-x is a 3′-blocking moiety to prevent extension from the 3′-terminus of the primer template.

### Claim 11 (depends on 9)

The method of claim 9, wherein the 3′-blocking moiety comprises 3′-TEG (triethylene glycol), 3′-SpC3 (1,3-propanediol), 3′-Sp18 (hexaethylene glycol), 3′-amino, 3′-phosphate, 3′-biotin, 3′-1′,2′-dideoxyribose, 3′-dideoxycytidine, or 3′-inverted deoxythymidine.

### Claim 12 (depends on 1)

The method of claim 1, wherein the primer template for the final primer extension reaction in step (f) comprises a poly A10-30 tail.

### Claim 13 (depends on 1)

The method of claim 1, wherein the primer template for the final primer extension reaction in step (f) comprises a poly N8-20 region.

### Claim 14 (depends on 1)

The method of claim 1, wherein the primer template for the final primer extension reaction in step (f) comprising a single stranded oligonucleotide having a structure comprising:
5′-A10-30-B-N8-20-Anchor N4-20-X-3′,
wherein:
A10-30 is a sequence of 10 to 30 deoxyadenosine residues;
B is a single deoxyguanosine, deoxycytidine, or deoxythymidine nucleotide;
N8-20 is a sequence of any 8 to 20 nucleotides;
Anchor N4-20 is an anchor sequence of 4 to 20 nucleotides; and
-x is a 3′-blocking moiety to prevent extension from the 3′-terminus of the primer template.

### Claim 15 (depends on 1)

The method of claim 1, wherein the primer template for the final primer extension reaction in step (f) comprising a single stranded oligonucleotide having a structure comprising:
5′-A10-30-B-N8-20-Anchor N8-20-X-3′,
wherein:
A10-30 is a sequence of 10 to 30 deoxyadenosine residues;
B is a single deoxyguanosine, deoxycytidine, or deoxythymidine nucleotide;
N8-20 is a sequence of any 8 to 20 nucleotides;
Anchor N8-20 is an anchor sequence of 8 to 20 nucleotides; and
-x is a 3′-blocking moiety to prevent extension from the 3′-terminus of the primer template.

### Claim 16 (depends on 1)

The method of claim 1, wherein the derivatization comprises hydroxyl, carboxyl, amine, aldehyde, or sulfate moieties for coupling with nucleic acids or modified nucleic acids.

### Claim 17 (depends on 16)

The method of claim 16, wherein the modified nucleic acid comprises an amino-terminated oligonucleotide.

### Claim 18 (depends on 1)

The method of claim 1, wherein the derivatized bead comprises natural or synthetic polymers or hydrogels, organic or inorganic particles, glass, ceramic, metal, paramagnetic particles, or combinations thereof.

### Claim 19 (depends on 1)

The method of claim 1, wherein the derivatized bead comprises poly(styrene-divinylbenzene) derivatized with hydroxyl moieties.

### Claim 20 (depends on 1)

The method of claim 1, wherein the substrate comprising one or more oligonucleotide templates has a concentration in the reaction of 10-40 mg/mL.

### Claim 21 (depends on 1)

The method of claim 1, wherein the reagents sufficient to perform an extension reaction comprise: a buffered solution, deoxyribonucleotide triphosphates (dNTPs), a DNA polymerase, and optionally, a pyrophosphatase.

### Claim 22 (depends on 21)

The method of claim 21, wherein the DNA polymerase is E. coli DNA polymerase Klenow fragment (Exo−).

### Claim 23 (depends on 21)

The method of claim 21, wherein the DNA polymerase is provided at a ratio of 1-10 U per nmol of oligonucleotide template.

### Claim 24 (depends on 21)

The method of claim 21, wherein the optional pyrophosphatase is provided at a ratio of 1 U pyrophosphatase per nmol of oligonucleotide template.

### Claim 25 (depends on 1)

The method of claim 1, wherein the period of time in step (d) comprises about 30 min to about 20 hours at a temperature of about 25° C. to about 37° C. with rotation of the extension reaction at 10-20 rpm.

### Claim 26 (depends on 1)

The method of claim 1, wherein the purification in step (e) comprises combining the substrates comprising the extension products together, washing the combination with hot water or buffer, and collecting the substrate comprising the primer extension product by centrifugation.

### Claim 27 (depends on 26)

The method of claim 26, wherein the purification step is performed at least 3 times.

### Claim 28 (depends on 1)

The method of claim 1, wherein following the purification in step (e), the substrates comprising the extension products are diluted and redistributed into individual reactions.

### Claim 29 (depends on 1)

The method of claim 1, wherein step (f) repeats steps (b) to (e) at least 3 to 100 times; each time using additional primer templates partially complementary to the extended product from each subsequent round.

### Claim 30 (depends on 1)

The method of claim 1, wherein step (f) repeats steps (b) to (e) at least 3 times, 4 times, 5, times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 50 times, 100 times, or even more each time using additional primer templates partially complementary to the extended product from each subsequent round.

### Claim 31 (depends on 1)

The method of claim 1, wherein the quantitating the amount of the oligonucleotide templates attached to the substrate, the amount of extension product attached to the substrate at each round, or the amount of the final extension product attached to the substrate comprises one or more of:
(a) calculating the concentration of the substrate comprising an oligonucleotide template, extension product, or final extension product in solution by diluting a solution of the substrate;
counting the number of substrates;
applying a dilution factor; and
obtaining a concentration of the substrate in solution;
or
(b) cleaving the oligonucleotide templates attached to the substrate, the amount of extension product attached to the substrate at each round, or the amount of the final extension product attached to the substrate with one or more enzymes that specifically cleave the phosphodiester linkage at deoxyuridine nucleotides; and
quantifying the amount of oligonucleotide templates attached to the substrate, the amount of extension product attached to the substrate at each round, or the amount of the final extension product attached to the substrate;
or
(e) diluting the substrate comprising the oligonucleotide template, the extension products after a round, or the final extension product;
preparing a serial dilution using control templates;
adding and annealing a fluorescently quenched probe oligonucleotide to the oligonucleotide template, the extension product, the final extension product, or the serially diluted control templates to obtain fluorescently labeled double stranded sequences;
measuring the fluorescence signal of the fluorescently labeled double stranded sequences;
preparing a standard curve of the fluorescence signal of the control templates; and
using the standard curve and the fluorescence signal of the of the oligonucleotide template, the extension product, or the final extension product to quantitate the amount (mass or moles) of the oligonucleotide template, the extension product, or the final extension product; or
a combination thereof.

### Claim 32 (depends on 31)

The method of claim 31, wherein the one or more enzymes in step (b) comprises a mixture of uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII.