IP Library › Granted Patent US 12,398,423
Granted Patent B2
US 12,398,423 · App. 18/520,654 · Granted Aug 26, 2025

Single cell nucleic acid detection and analysis

Inventors: Xiaoliang Sunney Xie (Lexington, MA); Katsuyuki Shiroguchi (Arlington, MA); Peter A. Sims (Cambridge, MA); Tony Z. Jia (Cambridge, MA)
Assignee: President and Fellows of Harvard College
C12Q1/6874C12N15/1065C12Q1/6853C12N2320/10
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Quick Facts
Patent No.
US 12,398,423
App. No.
18/520,654
Granted
Aug 26, 2025
Kind
B2
Abstract

Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.

Claims (32)

1. A method of determining copy number of a nucleic acid molecule in a sample including a plurality of nucleic acid molecules comprising

attaching a unique barcode sequence to substantially each of the plurality of nucleic acid molecules in the sample to produce a plurality of barcoded nucleic acid molecules,

amplifying the plurality of barcoded nucleic acid molecules in the sample to produce amplicons of the plurality of barcoded nucleic acid molecules,

sequencing each amplicon to identify an associated nucleic acid sequence and an associated barcode sequence,

selecting a first target nucleic acid sequence and determining the number of unique associated barcode sequences for the first target nucleic acid sequence, wherein the number of unique associated barcode sequences is the copy number of the first target nucleic acid sequence.

2. The method of claim 1 wherein the nucleic acid molecules are DNA or RNA.

3. The method of claim 1 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded RNA molecules and further including the steps of reverse transcribing the plurality of barcoded RNA molecules to produce barcoded cDNA molecules and amplifying the barcoded cDNA molecules to produce amplicons of the barcoded cDNA molecules.

4. The method of claim 3 comprising repeatedly reverse transcribing the plurality of barcoded RNA molecules to produce linear pre-amplified barcoded cDNA molecules and amplifying the linear pre-amplified barcoded cDNA molecules to produce amplicons of the linear pre-amplified barcoded cDNA molecules.

5. The method of claim 4 wherein the step of repeatedly reverse transcribing the plurality of barcoded RNA molecules includes using reverse transcriptase and a nicking enzyme.

6. The method of claim 1 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded DNA molecules and further including the steps of repeated replication of the plurality of barcoded DNA molecules to produce a plurality of pre-amplified barcoded DNA molecules and amplifying the plurality of pre-amplified barcoded DNA molecules to produce amplicons of the plurality of pre-amplified barcoded DNA molecules.

7. The method of claim 6 wherein the step of repeated replication of the plurality of barcoded DNA molecules includes using DNA polymerase and a nicking enzyme.

8. The method of claim 1 wherein the sample is obtained from one or more cells of a first cell type and wherein amplification includes use of a primer generated from genomic DNA of the first cell type.

9. A method of counting nucleic acid molecules in a sample including a plurality of nucleic acid molecules comprising

attaching a unique barcode sequence to substantially each of the plurality of nucleic acid molecules in the sample to produce a plurality of barcoded nucleic acid molecules,

amplifying the plurality of barcoded nucleic acid molecules in the sample to produce amplicons of the plurality of barcoded nucleic acid molecules,

sequencing each amplicon to identify an associated barcode sequence, and

counting the number of unique associated barcode sequences as a measure of the number of nucleic acid molecules in the sample.

10. The method of claim 9 wherein the nucleic acid molecules are DNA or RNA.

11. The method of claim 9 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded RNA molecules and further including the steps of reverse transcribing the plurality of barcoded RNA molecules to produce barcoded cDNA molecules and amplifying the plurality of barcoded cDNA molecules to produce amplicons of the plurality of barcoded cDNA molecules.

12. The method of claim 11 comprising repeatedly reverse transcribing the plurality of barcoded RNA molecules to produce linear pre-amplified barcoded cDNA molecules and amplifying the linear pre-amplified barcoded cDNA molecules to produce amplicons of the linear pre-amplified barcoded cDNA molecules.

13. The method of claim 12 wherein the step of repeatedly reverse transcribing the plurality of barcoded RNA molecules includes using reverse transcriptase and a nicking enzyme.

14. The method of claim 9 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded DNA molecules and further including the steps of repeated replication of the plurality of barcoded DNA molecules to produce a plurality of pre-amplified barcoded DNA molecules and amplifying the plurality of pre-amplified barcoded DNA molecules to produce amplicons of the plurality of pre-amplified barcoded DNA molecules.

15. The method of claim 14 wherein the step of repeated replication of the plurality of barcoded DNA molecules includes using DNA polymerase and a nicking enzyme.

16. The method of claim 9 wherein the sample is obtained from one or more cells of a first cell type and wherein amplification includes use of a primer generated from genomic DNA of the first cell type.

17. A method of determining copy numbers of nucleic acid molecules in a sample comprising

attaching a unique barcode sequence to substantially each of the nucleic acid molecules in the sample to produce a plurality of barcoded nucleic acid molecules,

amplifying the plurality of barcoded nucleic acid molecules in the sample to produce amplicons of the plurality of barcoded nucleic acid molecules,

massively parallel sequencing the amplicons of the plurality of barcoded nucleic acid molecules to identify for each amplicon an associated nucleic acid sequence and an associated barcode sequence, and

determining the number of unique associated barcode sequences for each nucleic acid sequence in the sample.

18. The method of claim 17 wherein the nucleic acid molecules are DNA or RNA.

19. The method of claim 17 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded RNA molecules and further including the steps of reverse transcribing the plurality of barcoded RNA molecules to produce barcoded cDNA molecules and amplifying the plurality of barcoded cDNA molecules to produce amplicons of the plurality of barcoded cDNA molecules.

20. The method of claim 19 comprising repeatedly reverse transcribing the plurality of barcoded RNA molecules to produce linear pre-amplified barcoded cDNA molecules and amplifying the linear pre-amplified barcoded cDNA molecules to produce amplicons of the linear pre-amplified barcoded cDNA molecules.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 20, 2025
From: JIA, TONY Z.; SHIROGUCHI, KATSUYUKI; SIMS, PETER A.; XIE, XIAOLIANG SUNNEY
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 072595/0637 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 11, 2025
From: JIA, TONY Z.; SHIROGUCHI, KATSUYUKI; SIMS, PETER A.; XIE, XIAOLIANG SUNNEY
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 070469/0556 →
Continuity (10)
Continuation 17320361 · May 14, 2021
Continuation 16847718 · Apr 14, 2020
Continuation 16774104 · Jan 28, 2020
Continuation 16364947 · Mar 26, 2019
Continuation 15730157 · Oct 11, 2017
Continuation 14990286 · Jan 7, 2016
Continuation 14006971
Provisional Application 61583787 · Jan 6, 2012
Provisional Application 61467037 · Mar 24, 2011
Related Publication 20240093294A1 · Mar 21, 2024
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