Granted Patent B2

US 12,630,864 · App. 17/746,191 · Granted May 19, 2026

Inventors: Joshua Stahl (Boulder, CO); Jason Myers (Boulder, CO); Brady Culver (Aliso Viejo, CA); Brian Kudlow (Boulder, CO); Jens Eberlein (Boulder, CO)

Assignee: Laboratory Corporation of America Holdings

C12Q1/6813A61P35/02C07K16/2809C12N15/09C12Q1/6806C07D495/04C07K2319/32C12Q1/6869

Abstract

Claims (36)

1 . A method of preparing nucleic acids for analysis, the method comprising:

(a) hybridizing a primer to a nucleic acid molecule comprising a target nucleotide sequence, wherein the nucleic acid molecule was obtained from cell-free DNA obtained from a subject having or suspected of having a cancer;

(b) generating a double stranded nucleic acid by a process comprising conducting a polymerase extension that is primed by the hybridized primer and that uses the nucleic acid molecule as a template, wherein the conducting of the polymerase extension comprises incorporating one or more capture moiety modified nucleotides into the double stranded nucleic acid, wherein each of the capture moiety modified nucleotides comprises a capture moiety;

(c) ligating an adapter nucleic acid to the double-stranded nucleic acid to produce a ligation product comprising the capture moiety;

(d) isolating the ligation product by a process comprising contacting the ligation product with a binding partner of the capture moiety; and

(e) amplifying the isolated ligation product by a polymerase chain reaction using (i) a first adapter primer that specifically anneals to a sequence of the adapter nucleic acid and (ii) a target-specific primer that comprises a 3′ portion that specifically anneals to the target nucleotide sequence and a 5′ tail portion that does not specifically anneal to the target nucleotide sequence, thereby providing a first amplification product.

2 . The method of claim 1 , further comprising:

(f) amplifying the first amplification product by a polymerase chain reaction using a tail primer that comprises a 3′ portion that specifically anneals to a complementary sequence of the 5′ tail portion of the target-specific primer and a second adapter primer that specifically anneals to a portion of the adapter nucleic acid, wherein the tail primer comprises a 5′ portion that does not specifically anneal to a complementary sequence of the target-specific primer.

3 . The method of claim 1 , wherein the one or more capture moiety modified nucleotides comprise biotin or a biotin moiety.

4 . The method of claim 3 , wherein the biotin moiety is selected from biotin-triethylene glycol, bis-biotin, photocleavable biotin, desthiobiotin, desthiobiotin-triethylene glycol, and biotin azide.

5 . The method of claim 3 , wherein the binding partner comprises streptavidin or streptavidin attached to a substrate.

6 . The method of claim 1 , wherein the nucleic acid molecule was derived from a cancer or tumor.

7 . The method of claim 1 , wherein the cell-free DNA comprises circulating tumor DNA.

8 . The method of claim 1 , wherein the cell free DNA was obtained from blood obtained from the subject.

9 . The method of claim 6 , wherein the cancer comprises a T cell malignancy or a B cell malignancy.

10 . The method of claim 1 , wherein the subject is a chordate or a human.

11 . The method of claim 2 , wherein:

the first adapter primer and the second adapter primer are different.

12 . The method of claim 11 , wherein the second adapter primer is nested relative to the first adapter primer.

13 . The method of claim 2 , wherein the 5′ portion of the tail primer comprises a sample index region, a molecular barcode region, or a sequence identical to a portion of a sequencing primer.

14 . The method of claim 2 , wherein the first adapter primer and the second adapter primer are the same, and the amplifying of (d) and the amplifying of (e) takes place in a same reaction mixture or at the same time.

15 . The method of claim 2 further comprising: after the amplifying of (f), determining a sequence of at least a portion of the target nucleic acid sequence.

16 . The method of claim 15 , further comprising detecting a tumor in the subject.

17 . The method of claim 1 , wherein the primer hybridized to the nucleic acid molecule in (a) comprises a capture moiety modified nucleotide.

18 . The method of claim 1 , wherein the primer that hybridizes to the nucleic acid molecule in (a) is a first target specific primer, or wherein the primer that hybridizes to the nucleic acid molecule in (a) is one of a population of random primers.

19 . The method of claim 1 , wherein the isolating of (d) comprises:

i) immobilizing the ligation product, which comprises the capture moiety, on a substrate comprising the binding partner;

ii) washing the immobilized ligation product; and

iii) releasing the washed ligation product from the substrate.

20 . A method of preparing nucleic acids for analysis, the method comprising:

(a) hybridizing a plurality of different target-specific primers to a population of nucleic acid molecules comprising a plurality of different target nucleotide sequences, wherein the population of nucleic acid molecules was obtained from cell-free DNA obtained from a human subject having or suspected of having a cancer;

(b) generating a plurality of double stranded nucleic acids by a process comprising conducting a polymerase extension that is primed by each of the plurality of different target-specific primers, wherein the conducting of the polymerase extension comprises incorporating one or more capture moiety modified nucleotides into the double stranded nucleic acids, and wherein each of the capture moiety modified nucleotides comprises a capture moiety;

(c) ligating an adapter nucleic acid to each of the plurality of double-stranded nucleic acids to produce a plurality of ligation products, each comprising at least one of the capture moiety;

(d) isolating the plurality of ligation products by a process comprising contacting the plurality of ligation products with a substrate or plurality of beads comprising a binding partner of the capture moiety;

(e) amplifying the isolated ligation products by a polymerase chain reaction using (i) a first adapter primer that specifically anneals to a sequence of the adapter nucleic acid and (ii) a plurality of different target-specific primers, each comprising a 3′ portion that specifically anneals to one of the plurality of different target nucleotide sequences and a substantially identical 5′ tail portion that does not specifically anneal to a target nucleotide sequence, thereby providing a plurality of first amplification products; and

(f) amplifying the plurality of first amplification products by a polymerase chain reaction using (i) a tail primer that comprises a 3′ portion that specifically anneals to a complementary sequence of the 5′ tail portion of the plurality of different target-specific primers and (ii) a second adapter primer that specifically anneals to a portion of the adapter nucleic acid, thereby providing a plurality of second amplification products.

Assignments (6)

Continuity (10)

References Cited (400)