IP Library › Granted Patent US 12,371,732
Granted Patent B2
US 12,371,732 · App. 18/481,021 · Granted Jul 29, 2025

Methods of preparing nucleic acids for sequencing

Inventors: Anthony John Iafrate (Newton, MA); Long Phi Le (Boston, MA); Zongli Zheng (Boston, MA); Jason Myers (Golden, CO); Joshua Stahl (Boulder, CO)
Assignees: The General Hospital Corporation; ARCHERDX, LLC
C12Q1/6806C12Q1/6809
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Quick Facts
Patent No.
US 12,371,732
App. No.
18/481,021
Granted
Jul 29, 2025
Kind
B2
Abstract

Aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.

Claims (23)

1. A method of determining a nucleotide sequence contiguous to a known target nucleotide sequence, the method comprising;

(a) contacting a target nucleic acid molecule comprising the known target nucleotide sequence and the nucleotide sequence contiguous to the known target nucleotide sequence with a first target primer under hybridization conditions;

(b) performing a template-dependent extension reaction that is primed by a first target primer that hybridizes to the known target nucleotide sequence;

(c) contacting an extension product of (b) with a population of tailed random primers under hybridization conditions, wherein each of the population of tailed random primers comprises a common sequence that is 5′ to a 3′ nucleic acid sequence that is 6 to 12 random nucleotides;

(d) performing a template-dependent extension reaction that is primed by at least one of the tailed random primers that hybridizes to a portion of the extension product of (b);

(e) amplifying the extension product of (d) using a second target primer, and a first tail primer comprising a 3′ sequence that specifically anneals to a complement of the common sequence; and

(f) determining a sequence of the nucleotide sequence contiguous to the known target nucleotide sequence by sequencing an amplicon of (e) using a first and/or second sequencing primer.

2. The method of claim 1 , wherein the population of tailed random primers comprises single-stranded oligonucleotide molecules, and the common sequence comprises a nucleic acid sequence identical to the first or second sequencing primer.

3. The method of claim 1 , wherein the second target primer comprises a 3′ portion comprising a nucleic acid sequence that can specifically anneal to a portion of the known target nucleotide sequence.

4. The method of claim 1 , wherein the second target primer is nested with respect to the first target primer.

5. The method of claim 1 , wherein the first tail primer comprises a nucleic acid sequence identical to a 5′ portion of the tailed random primer.

6. The method of claim 1 , wherein the sequencing of (g) comprises a next generation sequencing method.

7. The method of claim 1 , wherein the common sequence of each of the population of tailed random primers is substantially identical.

8. The method of claim 1 , wherein each of the population of tailed random primers comprises a unique barcode sequence.

9. The method of claim 1 , wherein each of the population of tailed random primers can form a hair-pin loop structure.

10. The method of claim 1 , wherein the first target primer and the second target primer are identical.

11. The method of claim 1 , wherein the first target primer or the second target primer comprise a sequence identical to the first or second sequencing primer.

12. The method of claim 1 , wherein the first tail primer or the second tail primer comprise a sequence identical to the first or second sequencing primer.

13. The method of claim 1 , wherein any unhybridized primers are removed from a reaction after an extension or amplification reaction.

14. The method of claim 1 , wherein the target nucleic acid molecule comprises a gene rearrangement.

15. The method of claim 14 , wherein the gene rearrangement is present in a nucleic acid selected from the group consisting of: genomic DNA; RNA; and cDNA.

16. The method of claim 15 , wherein the gene rearrangement comprises an oncogene.

17. The method of claim 1 , wherein the method comprises contacting the sample with a plurality of different first target primers and a plurality of different second target primers.

Assignments (6)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 17, 2025
From: ARCHERDX, LLC
To: INVITAE CORPORATION
Reel/Frame 073239/0615 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 30, 2024
From: INVITAE CORPORATION
To: LABORATORY CORPORATION OF AMERICA HOLDINGS
Reel/Frame 068822/0025 →
SECURITY INTEREST Recorded Jan 16, 2024
From: ARCHERDX, LLC
To: U.S. BANK TRUST COMPANY, NATIONAL ASSOCIATION
Reel/Frame 066137/0765 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 17, 2023
From: IAFRATE, ANTHONY JOHN; LE, LONG PHI; ZHENG, ZONGLI
To: THE GENERAL HOSPITAL CORPORATION
Reel/Frame 065602/0880 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 17, 2023
From: MYERS, JASON; STAHL, JOSHUA
To: ARCHERDX, INC.
Reel/Frame 065603/0060 →
CHANGE OF NAME Recorded Nov 17, 2023
From: ARCHERDX, INC.
To: ARCHERDX, LLC
Reel/Frame 065603/0082 →
Continuity (4)
Continuation 16590878 · Oct 2, 2019
Continuation 14605363 · Jan 26, 2015
Provisional Application 61931959 · Jan 27, 2014
Related Publication 20240158832A1 · May 16, 2024
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