IP Library › Granted Patent US 12,539,510
Granted Patent B2
US 12,539,510 · App. 17/586,645 · Granted Feb 3, 2026

Integrated purification and measurement of DNA methylation and co-measurement of mutations and/or mRNA expression levels in an automated reaction cartridge

Inventors: Edwin Wei-Lung Lai (Santa Clara, CA); Andrew Kohlway (Santa Clara, CA); Reuel Van Atta (Sunnyvale, CA); Russell Higuchi (Alameda, CA); Alexander A. Gall (Woodinville, WA); Kriszten Kocmond (Los Altos, CA)
Assignee: Cepheid
B01L3/502B01L7/52C12N15/1006C12Q1/6827C12Q1/6853C12Q1/686B01L3/5027B01L2200/16B01L2300/0816B01L2300/0861B01L2300/0864B01L2300/087B01L2300/1805B01L2400/0478B01L2400/0644C12Q2537/143C12Q2537/149C12Q2537/164
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Quick Facts
Patent No.
US 12,539,510
App. No.
17/586,645
Granted
Feb 3, 2026
Kind
B2
Abstract

Methods of determining methylation of DNA are provided that include filtering a biological sample comprising a nucleic acid with a first matrix material to purify the DNA; eluting and denaturing the DNA to produce eluted denatured DNA; heating the DNA in the presence of bisulfite ions to produce deaminated nucleic acid; optionally contacting said deaminated nucleic acid with a second matrix material; desulphonating and eluting the bound deaminated nucleic acid; and performing methylation specific PCR, nucleic acid sequencing, and/or high resolution melting analysis (HRM) on said bisulfite-converted nucleic acid to determine the methylation of said nucleic acid, wherein multiple steps may be performed in a single reaction cartridge.

Claims (51)

1 . A cartridge for determining a methylation state of a nucleic acid, said cartridge comprising:

(i) a first column comprising a first matrix material,

(ii) a sample receiving chamber,

(iii) a reaction channel or chamber that can be temperature controlled,

(iv) a plurality of chambers for containing reagents and/or buffers, wherein:

at least one of said chambers is configured to contain a bisulfite reagent, and

at least one of said chambers is configured to contain a desulphonation/elution buffer,

wherein said cartridge optionally comprises a second column comprising a second matrix material,

wherein said sample receiving chamber, said first and optional second column(s), said plurality of chambers, and said temperature-controlled heating channel or chamber, are selectively in fluid communication by microfluidic channels and valves, and wherein:

said sample receiving chamber, said first and optional second column(s), said plurality of chambers, and said reaction channel or chamber or a port into said reaction channel or chamber, are disposed around a central valve and selectively in fluid communication with a channel in said central valve, wherein said central valve is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with said central valve, and/or

said cartridge is configured to perform bisulfite conversion in the reaction channel or chamber, which can be subjected to thermocycling and later used for amplification of converted nucleic acid by polymerase chain reaction (PCR).

2 . The cartridge of claim 1 , wherein said sample receiving chamber, said first and optional second column(s), said plurality of chambers, and said reaction channel or chamber or a port into said reaction channel or chamber, are disposed around a central valve and selectively in fluid communication with a channel in said central valve, wherein said central valve is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with said central valve.

3 . The cartridge of claim 1 , wherein said cartridge is configured to perform bisulfite conversion in the reaction channel or chamber, which can be subjected to thermocycling and later used for amplification of converted nucleic acid by polymerase chain reaction (PCR).

4 . The cartridge of claim 1 , wherein:

said sample receiving chamber, said first and optional second column(s), said plurality of chambers, and said reaction channel or chamber or a port into said reaction channel or chamber, being disposed around a central valve and selectively in fluid communication with a channel in said central valve, wherein said central valve is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with said central valve, and

said cartridge is configured to perform bisulfite conversion in the temperature controlled channel or chamber, which can be subjected to thermocycling and later used for amplification of converted nucleic acid by polymerase chain reaction (PCR).

5 . The cartridge of claim 1 , wherein said cartridge comprises a chamber configured to contain a reagent comprising guanidinium thiocyanate ethanol (GTC-EtOH).

6 . The cartridge of claim 1 , wherein said second column is absent.

7 . The cartridge of claim 1 , wherein said reaction channel or chamber is a channel or chamber that can be subjected to thermocycling.

8 . The cartridge of claim 1 wherein said bisulfite reagent comprises a compound selected from the group consisting of ammonium bisulfite sodium, metabisulfite, potassium bisulfite, cesium bisulfite, and 1,4-diazoniabicyclo[2.2.2]octane-1,4-disulfinate (DABSO).

9 . The cartridge of claim 1 , wherein said cartridge comprises one, two, or more chambers containing one or more reagents selected from the group consisting of methylation specific PCR primers, methylation specific PCR probes, PCR enzyme(s), and PCR reaction buffer.

10 . The cartridge of claim 1 , wherein:

said cartridge contains at least one chamber containing primers and probes for detection of methylation of a forward strand of a converted DNA; and/or

said cartridge contains at least one chamber containing primers and probes for detection of methylation of a reverse strand of a converted DNA.

11 . The cartridge of claim 9 , wherein said PCR primers, and/or said probes, and/or enzymes are provided as beads.

12 . The cartridge of claim 1 , wherein said cartridge is configured to comprise:

a first chamber containing a sample, that optionally comprises a GTC-EtOH-Tween extraction/precipitation reagent;

a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH) solution;

a third chamber containing a bisulfite reagent;

a fourth chamber containing a buffer;

a fifth chamber containing a rinse solution; and

a sixth chamber containing an elution/desulphonation reagent.

13 . The cartridge of claim 1 , wherein:

the cartridge is configured for the bisulfite reagent to be added to the cartridge by the user; and/or

the cartridge is configured for addition of said GTC-ETOH-Tween buffer by the user.

14 . The cartridge of claim 1 , wherein:

the bisulfite reagent is provided as a component of the cartridge; and/or

the GTC-ETOH-Tween buffer is provided as a component of the cartridge.

15 . The cartridge of claim 12 , wherein said cartridge comprises a seventh chamber containing PCR primers and/or probes and/or PCR enzymes.

16 . The cartridge of claim 1 , wherein:

said cartridge comprises one or more chambers containing primers specific for bisulfite converted methylated and/or unmethylated sequences; and/or

said cartridge comprises one or more chambers containing reagents for TaqMan PCR reactions; and/or

said cartridge comprises one or more chambers containing one or more fluorescent probes that are markers for amplified methylated sequences and/or one or more fluorescent probes that are markers for amplified unmethylated sequences.

17 . The cartridge of claim 16 , wherein:

(a) said probes comprise a fluorescent reporter dye and a quencher dye, where the probes provide a signal upon cleavage by the 5′ to 3′ nuclease activity of Taq DNA polymerase; and/or

(b) said cartridge comprises a plurality of probes each specific to a different methylated region in an amplified region of interest; or said cartridge comprises a plurality of probes each specific to the same methylated region in an amplified region of interest; or said cartridge comprises a single probe specific to a methylated region in an amplified region of interest; and/or

(c) said cartridge contains primers and/or probes to determine methylation of a promoter region of a gene selected from the group consisting of MGMT, RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, and AKR1B1; and/or said cartridge contains one or more primers shown in Tables 5, 9, or 10, and/or one or more probes shown in Tables 5, 9, or 10; and/or

(d) the cartridge is configured for determination of the expression level of RNA for a methyltransferase.

18 . The cartridge of claim 16 , wherein said probes comprise a fluorescent reporter dye and a quencher dye, where the probes provide a signal upon cleavage by the 5′ to 3′ nuclease activity of Taq DNA polymerase.

19 . The cartridge of claim 16 , wherein said cartridge comprises a plurality of probes each specific to a different methylated region in an amplified region of interest; or said cartridge comprises a plurality of probes each specific to the same methylated region in an amplified region of interest; or said cartridge comprises a single probe specific to a methylated region in an amplified region of interest.

20 . The cartridge of claim 16 , wherein said cartridge contains primers and/or probes to determine methylation of a promoter region of a gene selected from the group consisting of MGMT, RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, and AKR1B1; and/or said cartridge contains one or more primers shown in Tables 5, 9, or 10, and/or one or more probes shown in Tables 5, 9, or 10.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 7, 2024
From: LAI, EDWIN WEI-LUNG; KOHLWAY, ANDREW; VAN ATTA, REUEL; HIGUCHI, RUSSELL; GALL, ALEXANDER A.; KOCMOND, KRISZTEN
To: CEPHEID
Reel/Frame 068817/0659 →
Continuity (3)
Continuation 15839731 · Dec 12, 2017
Provisional Application 62433165 · Dec 12, 2016
Related Publication 20220226809A1 · Jul 21, 2022
References Cited (111)
US 5958349A · Petersen et al. · 1999 [cited by applicant]
US 6403037B1 · Chang et al. · 2002 [cited by applicant]
US 6440725B1 · Pourahmadi et al. · 2002 [cited by applicant]
US 6783736B1 · Taylor et al. · 2004 [cited by applicant]
US 6818185B1 · Petersen et al. · 2004 [cited by applicant]
US 8062849B2 · Sukumar et al. · 2011 [cited by applicant]
US 10450609B2 · Sukumar et al. · 2019 [cited by applicant]
US 10533210B2 · Nauwelaers et al. · 2020 [cited by applicant]
US 11260387B2 · Lai et al. · 2022 [cited by applicant]
US 20030082600A1 · Olek et al. · 2003 [cited by applicant]
US 20060068399A1 · McMillan et al. · 2006 [cited by applicant]
US 20060134643A1 · Berlin et al. · 2006 [cited by applicant]
US 20060286577A1 · Jia · 2006 [cited by applicant]
US 20070098600A1 · Kayyem · 2007 [cited by applicant]
US 20080014576A1 · Jovanovich et al. · 2008 [cited by applicant]
US 20090036665A1 · Domingo et al. · 2009 [cited by applicant]
US 20090253181A1 · Vangbo et al. · 2009 [cited by applicant]
US 20100041024A1 · Fuhrmann et al. · 2010 [cited by applicant]
US 20100330580A1 · Gimenez et al. · 2010 [cited by applicant]
US 20120088249A1 · Jovanovich et al. · 2012 [cited by applicant]
US 20120252015A1 · Hindson et al. · 2012 [cited by applicant]
US 20140098252A1 · Chang et al. · 2014 [cited by applicant]
US 20140272967A1 · Gundling et al. · 2014 [cited by applicant]
US 20140272997A1 · Ivie et al. · 2014 [cited by applicant]
US 20140274735A1 · Granados · 2014 [cited by applicant]
US 20150099670A1 · Li et al. · 2015 [cited by applicant]
US 20150119268A1 · Bishop · 2015 [cited by applicant]
US 20150204813A1 · Toumazou et al. · 2015 [cited by applicant]
US 20160223442A1 · Guldberg et al. · 2016 [cited by applicant]
US 20170137871A1 · Lai et al. · 2017 [cited by applicant]
US 20180214864A1 · Lai et al. · 2018 [cited by applicant]
CN 101285096A · 2008 [cited by applicant]
CN 101984069A · 2011 [cited by applicant]
CN 104321442A · 2015 [cited by applicant]
CN 105378108A · 2016 [cited by applicant]
CN 107922941A · 2018 [cited by applicant]
EP 1394173A1 · 2004 [cited by applicant]
EP 2218793A1 · 2010 [cited by applicant]
JP 2009519023A · 2009 [cited by applicant]
JP 2009236933A · 2009 [cited by applicant]
RU 2332462C2 · 2008 [cited by applicant]
WO WO9856952A1 · 1998 [cited by applicant]
WO WO9920396A1 · 1999 [cited by examiner]
WO WO9933559A1 · 1999 [cited by applicant]
WO WO0073412A2 · 2000 [cited by applicant]
WO WO2004096825A1 · 2004 [cited by applicant]
WO WO2006136990A2 · 2006 [cited by applicant]
WO WO2007004103A1 · 2007 [cited by applicant]
WO WO2007068437A1 · 2007 [cited by applicant]
WO WO2009024019A1 · 2009 [cited by applicant]
WO WO2014052551A1 · 2014 [cited by applicant]
WO WO2016205233A2 · 2016 [cited by applicant]
WO WO2018111935A1 · 2018 [cited by applicant]
Analytic Jena AG., (2014) “innuCONVERT Bisulfite All-in-One Kit”, AJ Innuscreen Gmbh, 4 pages, Retrieved from the Internet: URL: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093933 [Retrieved on Nov… [cited by applicant]
Athamanolap et al. (2014) “Droplet Array Platform for High-Resolution Melt Analysis of DNA Methylation Density” Journal of Laboratory Automation 19(3): 304-312. [cited by applicant]
AU First Office Action dated Aug. 26, 2021 issued in AU 2016277943. [cited by applicant]
AU Office Action dated Feb. 1, 2022 in Application No. AU2017376118. [cited by applicant]
Bailey et al. (2010) “Single-Tube Analysis of DNA Methylation with Silica Superparamagnetic Beads” Clinical Chemistry, 56(6): 1022-1025. [cited by applicant]
Bianco et al. (1999) “Methylation-sensitive, single-strand conformation analysis (Ms-Ssca): A rapid method to screen for and analyze methylation” Hum. Mutat. 14(4): 289-293. [cited by applicant]
BR Office Action dated Aug. 3, 2021 issued in BR 112019011670-2. [cited by applicant]
Butkus, Ben (2015) “Hopkins Lab, Cepheid Developing Methylated DNA Panel for Breast Cancer Dx, Monitoring GenomeWeb” Genomeweb Retrieved from the Internet: URL:https://www.genomeweb.com/molecular-diagnostics/hopkins-lab… [cited by applicant]
Clark, S. et al., “High Sensitivity mapping of methylated cytosines” Nucleic Acid Research, 1994, vol. 22, No. 15, pp. 2990-2997. [cited by applicant]
CN First Office Action with Search Report dated Jan. 14, 2021 issued in CN 201680048155.X. [cited by applicant]
CN First Office Action with Search Report dated Mar. 12, 2021 issued in CN 201780085871.X. [cited by applicant]
CN Office Action dated Feb. 8, 2022, in Application No. CN201780085871.X with English translation. [cited by applicant]
CN Office Action dated Nov. 26, 2021, in Application No. CN201680048155.X with English translation. [cited by applicant]
Colella et al. (2003) “Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites.” BioTechniques 35(1): 146-150. [cited by applicant]
Eads et al. (1999) “CpG Island Hypermethylation in Human Colorectal Tumors Is Not Associated with DNA Methyltransferase Overexpression” Cancer Res., 59: 2302-2306. [cited by applicant]
Eads et al. (2000) “MethyLight: a high-throughput assay to measure DNA methylation” Nucleic Acids Research, 28(8): e32 (9 pages). [cited by applicant]
Ehrich et al. (2005) “Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry.” Proc. Natl. Acad. Sci. USA, 102 (44): 15785-15790. [cited by applicant]
EP Extended Search Report dated May 27, 2021 issued in EP 20202659.7. [cited by applicant]
EP Office Action dated May 21, 2019 issued in EP 16732159.5. [cited by applicant]
EP Office Action dated Nov. 5, 2020 issued in EP 17822953.0. [cited by applicant]
Esteller et al. (1999) “Detection of Aberrant Promoter Hypermethylation of Tumor Suppressor Genes in Serum DNA from Non-Small Cell Lung Cancer Patients” Cancer Research, 59: 67-70. [cited by applicant]
Fink et al. (1998) “Real-time quantitative RT-PCR after laser-assisted cell picking.” Nat. Med., 4(11): 1329-1333. [cited by applicant]
Frommer et al. (1992) “A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.” Proc. Natl. Acad. Sci. USA, 89 (5): 1827-1831. [cited by applicant]
Herman et al. (1996) “Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.” Proc. Natl. Acad. Sci. USA, 93: 9821-9826. [cited by applicant]
Holmes, E.E. et al., “Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine”, 2014, PLOS One, 9(4): e93933 (14 pag… [cited by applicant]
IN Office Action dated Dec. 10, 2021, in Application No. IN201817001016. [cited by applicant]
JP Final Office Action dated Jun. 21, 2021 issued in JP 2017-564901. [cited by applicant]
JP Office Action dated Dec. 6, 2021, in Application No. JP2019-531197 with English translation. [cited by applicant]
JP Office Action dated Jul. 13, 2020 issued in JP 2017-564901. [cited by applicant]
Lee et al. (1993) “Allelic discrimination by nick-translation PCR with fluorogenic probes.” Nucleic Acids Res., 21(16): 3761-3766. [cited by applicant]
Livak et al. (1995) “Oligonucleotides With Fluorescent Dyes at Opposite Ends Provide a Quenched Probe System Useful for Detecting PCR Product and Nucleic Acid Hybridization” Genome Res. PCR Meth. Appl., 4: 357-362. [cited by applicant]
Meissner, A. et al., “Reduced Representation Bisulfite Sequencing for Comparative High-resolution DNA Methylation Analysis”, Nucleic Acid Research, 2005, vol. 33, No. 18, pp. 5868-5877. [cited by applicant]
Notice of Allowance dated Oct. 29, 2021 in U.S. Appl. No. 15/839,731. [cited by applicant]
PCT International Preliminary Report on Patentability dated Dec. 19, 2017 issued in PCT/US2016/037422. [cited by applicant]
PCT International Preliminary Report on Patentability dated Jun. 18, 2019 issued in PCT/US2017/065905. [cited by applicant]
PCT International Search Report and Written Opinion dated Apr. 3, 2018 issued in PCT/US2017/065905. [cited by applicant]
PCT International Search Report and Written Opinion dated Dec. 6, 2016 issued in PCT/US2016/037422. [cited by applicant]
PCT Invitation to pay additional fees dated Oct. 4, 2016 issued in PCT/US2016/037422. [cited by applicant]
Qian and Brent (1997) “Methylation Hot Spots in the 5′ Flanking Region Denote Silencing of the O6-Methylguanine-DNA Methyltransferase Gene” Cancer Research, 57: 3672-3677. [cited by applicant]
Rand et al. (2002) “Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives” Methods 27(2): 114-120. [cited by applicant]
Roche Diagnostics K.K, (Jan. 2005) “High Pure PCR Product Purification Kit,” 3 pages. [cited by applicant]
RU Office Action dated Dec. 8, 2021, in Application No. RU20190119086 with English Translation. [cited by applicant]
RU Office Action dated May 7, 2021, in Application No. RU20190119086 with English Translation. [cited by applicant]
Shin et al. (2013) “Droplet Bisulfite Conversion Platform for Epigenetic Cancer Biomarker Detection” Transducers & Eurosensors XXVII: The 17th International Conference on Solid-State Sensors, Actuators and Microsystems,… [cited by applicant]
Sommer and Tautz (1989) “Minimal homology requirements for PCR primers” Nucleic Acids Research, 17(16): 6749. [cited by applicant]
Tost et al. (2003) “Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing™” BioTechniques 35(1): 152-154 & 156. [cited by applicant]
Uccella et al. (2009) “MGMT methylation in diffuse large B-cell lymphoma: validation of quantitative methylation-specific PCR and comparison with MGMT protein expression” J Clin Pathol, 62:715-723. [cited by applicant]
US Corrected Notice of Allowability dated Sep. 24, 2021, in U.S. Appl. No. 15/839,731. [cited by applicant]
US Final Office Action dated Aug. 18, 2020 issued in U.S. Appl. No. 15/182,394. [cited by applicant]
US Final Office Action dated Jan. 28, 2020 issued in U.S. Appl. No. 15/182,394. [cited by applicant]
US Final Office Action dated Mar. 30, 2021 issued in U.S. Appl. No. 15/182,394. [cited by applicant]
U.S. Non-Final Office Action dated Nov. 22, 2021, in U.S. Appl. No. 15/182,394. [cited by applicant]
US Notice of Allowance dated Jun. 21, 2021 issued in U.S. Appl. No. 15/839,731. [cited by applicant]
US Office Action dated Jun. 4, 2018 issued in U.S. Appl. No. 15/182,394. [cited by applicant]
US Office Action dated Oct. 23, 2020 issued in U.S. Appl. No. 15/839,731. [cited by applicant]
Warnecke et al. (1997) “Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA.” Nucleic Acids Res., 25(21): 4422-4426. [cited by applicant]
Watts et al. (1997) “Methylation of Discrete Regions of the O6-Methylguanine DNA Methyltransferase (MGMT) CpG Island Is Associated with Heterochromatinization of the MGMT Transcription Start Site and Silencing of the Ge… [cited by applicant]
Wojdacz and Dobrovic (2007) “Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation” Nucleic Acids Res. 35(6): e41 (7 pages). [cited by applicant]