Granted Patent B2

US 12,203,127 · App. 15/727,887 · Granted Jan 21, 2025

Inventors: Christopher K. Raymond (Seattle, WA); Lee P. Lim (Kirkland, WA); Christopher D. Armour (Kirkland, WA)

C12Q1/6806C12Q1/6869G16B20/00G16B20/10G16B20/20G16B25/10G16B30/00G16B30/10G16B25/00

Abstract

Claims (34)

1. A method for targeted genetic analysis of circulating cell-free DNA (cfDNA), said method comprising:

(a) hybridizing one or more capture probe modules to a target genetic locus in a tagged cfDNA library to form one or more tagged cfDNA-capture probe module complexes,

wherein each capture probe module comprises a capture probe sequence at a 3′ end of the capture probe module and a tail sequence at a 5′ end of the capture probe module,

wherein the capture probe sequence is less than 60 nucleotides in length and hybridizes to a specific DNA target region,

wherein the one or more capture probe modules is not immobilized to a surface,

wherein the tail sequence comprises a primer binding site that is hybridized to a partner oligonucleotide comprising at its 3′ end a first member of a binding pair to allow for isolation and/or purification of a tagged cfDNA-capture probe module complex, and

wherein the tagged cfDNA library comprises cfDNA molecules ligated at each end to an adaptor;

(b) isolating the one or more tagged cfDNA capture probe module complexes from (a) to form one or more isolated tagged cfDNA-capture probe module complexes, wherein the isolating comprises binding the first member of the binding pair of a partner oligonucleotide hybridized to a tagged cfDNA-capture probe module complex to a second member of the binding pair, and each isolated tagged cfDNA capture probe module complex comprises a tagged cfDNA molecule and a capture probe module;

(c) enzymatically processing the one or more isolated tagged cfDNA-capture probe module complexes from (b) to generate one or more tagged hybrid nucleic acid molecules,

wherein the enzymatic processing of each isolated tagged cfDNA-capture probe module complex comprises performing 5′ to 3′ DNA polymerase extension of the capture probe module using the tagged cfDNA molecule as a template,

wherein each tagged hybrid nucleic acid molecule comprises the capture probe module and a complement of the tagged cfDNA molecule, and wherein the complement of the tagged cfDNA molecule comprises the complement of the adaptor;

(d) amplifying the one or more tagged hybrid nucleic acid molecules of (c) to generate amplified tagged hybrid nucleic acid molecules; and

(e) performing a quantitative genetic analysis of the amplified tagged hybrid nucleic acid molecules.

2. The method of claim 1 , wherein the primer binding site is a first primer binding site, and the adaptor comprises:

(a) a second primer binding site,

(b) a unique read code that identifies each unique sequencing read,

(c) one or more sequences for DNA sequencing; and, optionally,

(d) one or more sample codes for sample multiplexing.

3. The method of claim 1 , wherein the targeted genetic analysis is performed on a plurality of target genetic loci.

4. The method of claim 1 , wherein the capture probe sequences are about 25 nucleotides to about 45 nucleotides in length.

5. The method of claim 1 , wherein the capture probe sequences are 40 nucleotides in length.

6. The method of claim 1 , further comprising determining a number of genome equivalents in the tagged cfDNA library.

7. The method of claim 1 , wherein the tagged cfDNA library comprises cfDNA isolated from a biological sample of a subject selected from the group consisting of amniotic fluid, blood, plasma, serum, semen, lymphatic fluid, cerebral spinal fluid, ocular fluid, urine, saliva, stool, mucous, and sweat.

8. The method of claim 1 , wherein the quantitative genetic analysis comprises sequencing of the amplified tagged hybrid nucleic acid molecules to generate a plurality of sequencing reads and performing bioinformatics analysis on the plurality of sequencing reads.

9. The method of claim 8 , wherein the bioinformatics analysis is used to detect one or more genetic lesions with the target genetic locus.

10. The method of claim 9 , wherein the one or more genetic lesions comprise a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, or a change in copy number.

11. The method of claim 1 , wherein the quantitative genetic analysis is used to identify or detect one or more genetic lesions that cause or are associated with a genetic disease.

12. The method of claim 11 , wherein the genetic disease is cancer.

13. The method of claim 12 , wherein the one or more genetic lesions are chromosomal rearrangements.

14. The method of claim 11 , wherein the one or more genetic lesions comprise a genomic rearrangement that fuses a 3′ coding region of an ALK gene to another gene.

15. The method of claim 14 , wherein the 3′ coding region of the ALK gene is fused to an EML4 gene.

16. The method of claim 1 , wherein the first member of the binding pair comprises biotin.

17. The method of claim 1 , wherein a plurality of capture probe modules hybridize to the target genetic locus.

18. The method of claim 17 , wherein each of the plurality of capture probe modules hybridizes to the target genetic locus within about 200 bp of any other capture probe module.

Assignments (3)

Continuity (2)

References Cited (338)