US 12,404,556 B2Granted

Compositions and methods for detecting circulating tumor DNA

AssigneeQuest Diagnostics Investments Incorporated

InventorsSuzzette Arnal, Taraneh Angeloni

Life of this patent

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Abstract

The present technology provides polynucleotide compositions and methods of using the same to detect circulating tumor DNA (ctDNA) in a patient. Kits for use in practicing the methods are also provided.

Claims15

Dependent claims

A method for detecting at least one mutation in a double-stranded circulating tumor DNA (ctDNA) molecule present in a sample obtained from a patient comprising

(a) ligating a plurality of Y-shaped adapters to both ends of the double-stranded ctDNA molecule to form a double-stranded adapter-ctDNA complex, each Y-shaped adapter comprising a first oligonucleotide strand and a second oligonucleotide strand;
(b) amplifying both strands of the adapter-ctDNA complex to produce first amplicons and second amplicons, wherein the first amplicons are derived from the first oligonucleotide strand, and the second amplicons are derived from the second oligonucleotide strand;
(c) sequencing the first and second amplicons;
and
(d) detecting at least one mutation in the double-stranded ctDNA molecule, when a mutation detected in the first amplicons is consistent with a mutation detected in the second amplicons,
wherein the first oligonucleotide strand:
(i) comprises a first proximal region and a first distal region, wherein the first proximal region comprises a first unique molecular identifier sequence and a first spacer sequence having the sequence 5′ TGACT 3′ (SEQ ID NO:
49), wherein the first spacer sequence is located 3′ to the first unique molecular identifier sequence and wherein the “T” nucleotide located at the 3′ end of the first spacer sequence (SEQ ID NO:
49) contains a phosphorothioate bond;
and
(ii) does not comprise a degenerate or semi-degenerate sequence,
wherein the second oligonucleotide strand
(i) comprises a second proximal region and a second distal region, wherein the second proximal region comprises a second unique molecular identifier sequence and a second spacer sequence having the sequence 5′ GTCA 3′ (SEQ ID NO:
50), wherein the spacer sequence is located 5′ to the second unique molecular identifier;
and
(ii) does not comprise a degenerate or semi-degenerate sequence,
wherein the first proximal region of the first oligonucleotide strand hybridizes with the second proximal region of the second oligonucleotide strand,
wherein the first distal region of the first oligonucleotide strand does not hybridize with the second distal region of the second oligonucleotide strand,
wherein the second oligonucleotide strand comprises a nucleic acid sequence selected from the group consisting of:
(SEQ ID NO:
2)
5′ GTCAGCTACTGCAGCTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
4)
5′ GTCAGTATCATCATCAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
6)
5′ GTCACTCGACAGTCGAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
8)
5′ GTCATAGCTAGAGTACAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
10)
5′ GTCAACGAGTGCTCTGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
12)
5′ GTCAGACTATCGCATGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
14)
5′ GTCACTCGACTGATGAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
16)
5′ GTCAATACCGCTGATTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
18)
5′ GTCACAGTAGTATGCTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
20)
5′ GTCAACGTGTATCAGCAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
22)
5′ GTCACGTGTGACAGAGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
24)
5′ GTCATGATGACGTAGCAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
26)
5′ GTCAAGTGACATCTGCAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
28)
5′ GTCACTAGCTGTGAGTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
30)
5′ GTCATACATGAGCGAGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
32)
5′ GTCACTAGTGCAGCTAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
34)
5′ GTCATAGCTCGAACTGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
36)
5′ GTCAGCGAGTCATGCAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
38)
5′ GTCATGTACAGTACACAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
40)
5′ GTCATCAGACTCTAGTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
42)
5′ GTCAGACACGCACTCTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
44)
5′ GTCAATCTGATGCGTAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
46)
5′ GTCAACTGTCATGCAGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
and
(SEQ ID NO:
48)
5′ GTCAGTGAGATCGTACAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′.
and
wherein the Y-shaped adapters ligate to cell free DNA (cfDNA) at a concentration of as low as 5 ng.

115 more elements

Dependencies are inferred from the claim text.

Facts

Application: 17/819,824Filed 15 Aug 2022 · continuation
Priority: 28 Dec 2016
Published: US 2023/0061928 A12 Mar 2023
Granted: 2 Sep 2025
Term ends: Not estimatedThe source records don’t support an estimate.

Classification10 codes

C12Q 1/6886
For diseases caused by alterations of genetic material: for cancer
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6855
Using modified primers or templates: ligating adaptors
C12Q 2525/101
Modifications characterised by: incorporating non-naturally occurring nucleotides, e.g. inosine

Family

Application62/439,574Us provisional application · filed 28 Dec 2016
PCT/US2017/068483Parent of this continuation · filed 27 Dec 2017
US 2023/0061928 A1Pre-grant publication · published 2 Mar 2023
Application16/474,533Parent of this continuation
US 11,414,710 B2Parent of this continuation
US 2019/0330704 A1Published application
US 12,404,556 B2This patent

From the US filing history. Foreign family members aren’t listed.

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Abstract

A method for detecting at least one mutation in a double-stranded circulating tumor DNA (ctDNA) molecule present in a sample obtained from a patient comprising

(a) ligating a plurality of Y-shaped adapters to both ends of the double-stranded ctDNA molecule to form a double-stranded adapter-ctDNA complex, each Y-shaped adapter comprising a first oligonucleotide strand and a second oligonucleotide strand;
(b) amplifying both strands of the adapter-ctDNA complex to produce first amplicons and second amplicons, wherein the first amplicons are derived from the first oligonucleotide strand, and the second amplicons are derived from the second oligonucleotide strand;
(c) sequencing the first and second amplicons;
and
(d) detecting at least one mutation in the double-stranded ctDNA molecule, when a mutation detected in the first amplicons is consistent with a mutation detected in the second amplicons,
wherein the first oligonucleotide strand:
(i) comprises a first proximal region and a first distal region, wherein the first proximal region comprises a first unique molecular identifier sequence and a first spacer sequence having the sequence 5′ TGACT 3′ (SEQ ID NO:
49), wherein the first spacer sequence is located 3′ to the first unique molecular identifier sequence and wherein the “T” nucleotide located at the 3′ end of the first spacer sequence (SEQ ID NO:
49) contains a phosphorothioate bond;
and
(ii) does not comprise a degenerate or semi-degenerate sequence,
wherein the second oligonucleotide strand
(i) comprises a second proximal region and a second distal region, wherein the second proximal region comprises a second unique molecular identifier sequence and a second spacer sequence having the sequence 5′ GTCA 3′ (SEQ ID NO:
50), wherein the spacer sequence is located 5′ to the second unique molecular identifier;
and
(ii) does not comprise a degenerate or semi-degenerate sequence,
wherein the first proximal region of the first oligonucleotide strand hybridizes with the second proximal region of the second oligonucleotide strand,
wherein the first distal region of the first oligonucleotide strand does not hybridize with the second distal region of the second oligonucleotide strand,
wherein the second oligonucleotide strand comprises a nucleic acid sequence selected from the group consisting of:
(SEQ ID NO:
2)
5′ GTCAGCTACTGCAGCTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
4)
5′ GTCAGTATCATCATCAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
6)
5′ GTCACTCGACAGTCGAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
8)
5′ GTCATAGCTAGAGTACAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
10)
5′ GTCAACGAGTGCTCTGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
12)
5′ GTCAGACTATCGCATGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
14)
5′ GTCACTCGACTGATGAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
16)
5′ GTCAATACCGCTGATTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
18)
5′ GTCACAGTAGTATGCTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
20)
5′ GTCAACGTGTATCAGCAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
22)
5′ GTCACGTGTGACAGAGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
24)
5′ GTCATGATGACGTAGCAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
26)
5′ GTCAAGTGACATCTGCAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
28)
5′ GTCACTAGCTGTGAGTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
30)
5′ GTCATACATGAGCGAGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
32)
5′ GTCACTAGTGCAGCTAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
34)
5′ GTCATAGCTCGAACTGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
36)
5′ GTCAGCGAGTCATGCAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
38)
5′ GTCATGTACAGTACACAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
40)
5′ GTCATCAGACTCTAGTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
42)
5′ GTCAGACACGCACTCTAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
44)
5′ GTCAATCTGATGCGTAAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
(SEQ ID NO:
46)
5′ GTCAACTGTCATGCAGAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′;
and
(SEQ ID NO:
48)
5′ GTCAGTGAGATCGTACAGATCGGAAGAGCACACGTCTGAACTCCAGT
CAC 3′.
and
wherein the Y-shaped adapters ligate to cell free DNA (cfDNA) at a concentration of as low as 5 ng.

115 more elements

Facts

Application

17/819,824Filed 15 Aug 2022 · continuation

Priority

28 Dec 2016

Published

US 2023/0061928 A12 Mar 2023

Granted

2 Sep 2025

Term ends

Not estimatedThe source records don’t support an estimate.

Classification10 codes

C12Q 1/6886
For diseases caused by alterations of genetic material: for cancer
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6855
Using modified primers or templates: ligating adaptors
C12Q 2525/101
Modifications characterised by: incorporating non-naturally occurring nucleotides, e.g. inosine

Family

Application62/439,574Us provisional application · filed 28 Dec 2016

PCT/US2017/068483Parent of this continuation · filed 27 Dec 2017

US 2023/0061928 A1Pre-grant publication · published 2 Mar 2023

Application16/474,533Parent of this continuation

US 11,414,710 B2Parent of this continuation

US 2019/0330704 A1Published application

US 12,404,556 B2This patent

From the US filing history. Foreign family members aren’t listed.