(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`
`
`(43) International Publication Date
`17 February 2005 (17.02.2005)
`
`(10) International Publication Number
`
`WO 2005/014850 A2
`
`(51) International Patent Classification7:
`
`C12Q
`
`(21) International Application Number:
`PCT/US2004/025526
`
`(22) International Filing Date:
`
`6 August 2004 (06.08.2004)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/493,238
`60/568,958
`
`6 August 2003 (06.08.2003)
`7 May 2004 (07.05.2004)
`
`US
`US
`
`(71) Applicant (for all designated States except US): UNI-
`VERSITY OF MASSACHUSETTS [US/US]; One
`Beacon Street, 26th Floor, Boston, MA 02108 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): GINNS, Edward, I.
`[US/US]; 6 Woodchuck Hill Road, Shrewsbury, MA 01545
`(US). GALDZICKA, Marlena [US/US]; 2 Golden Hill
`Avenue, Shrewsbury, MA 01545 (US).
`
`(81) Designated States (unless otherwise indicated, for ever
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
`MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM, PG,
`PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SY, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM,
`ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARlPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl,
`FR, GB, GR, HU, IE, IT, LU, MC, NL, PL, PT, RO, SE, SI,
`SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, 'l‘G).
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report
`
`(74) Agent: GARCIA, Todd, E.; Fish & Richardson PC, 225
`Franklin Street, Boston, MA 02110—2804 (US).
`
`For two—letter codes and other abbreviations, refer to the ”Guid—
`ance Notes on Codes and Abbreviations ” appearing at the begin—
`ning ofeach regular issue ofthe PCT Gazette.
`
`(54) Title: SYSTEMS AND METHODS FOR ANALYZING NUCLEIC ACID SEQUENCES
`
`(57) Abstract: The invention relates to systems and methods for analyzing clinically relevant nucleic acid sequences.
`
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`WO 2005/014850
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`PCT/US2004/025526
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` Systems and Methods forxAnalyzing Nucleic Acid Sequences
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`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`This application claims priority to US. Provisional Application Nos. 60/493,238, filed on
`
`August 6, 2003, and 60/568,958, filed on May 7, 2004. The contents of both of those provisional
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`applications is incorporated herein by reference in its entirety.
`
`This invention relates to systems and methods for analyzing clinically relevant nucleic
`
`TECHNICAL FIELD
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`acid sequences.
`
`BACKGROUND
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`The healthcare delivery system has changed remarkably over the past several decades.
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`Clinical laboratories are under increasing pressure to deliver low cost and highly accurate
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`analytical services with the rapid tum—around time required by physicians and patients.
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`Laboratory testing has changed and improved in recent years to meet the challenge. Robotics
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`has been introduced to the laboratory to increase efficiency and reduce the need for human
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`participation, and laboratory instruments have been designed to decrease the biological sample
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`volumes needed to perform various assays. However, more improvement in the clinical
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`laboratory area is required to meet the demands of the ever-changing healthcare system.
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`SUMMARY
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`The present invention provides novel automated systems and methods to perform assays
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`on nucleic acid sequences (e.g., clinically relevant nucleic acid sequences). The system can
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`provide assay results quickly, accurately, and in a format easily accessible by health care
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`providers and/or third party payorsu(e.g., insurance companies). The invention also provides
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`novel and highly accurate assays using mass spectrometry (e. g., matrix—assisted laser
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`desorption/ionization (MALDD).
`
`In one aspect, the invention provides a system for performing a diagnostic assay on a
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`biological sample. The system includes, as its main components (a) a central controller
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`programmed to: (i) exchange information about the biological sample with an outside system or
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`database; and (ii) exchange information about the biological sample with one or more modules of
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`the system; (b) a sample transfer module for transferring a portion of the sample to a first
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`container; (0) a nucleic acid extraction module for extracting nucleic acids from cells within the
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`portion and for transferring the portion from the first container to a second container; ((1) a
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`nucleic acid measurement module for measuring the concentration of nucleic acids in the
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`portion; (6) a PCR preparation module for adding polymerase chain reaction (PCR) reaction
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`materials (e. g., individual nucleotides, primers, polymerase enzymes, and reagents) to the
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`portion; (1) a therrnocyling module for amplifying a target sequence and extending a primer in
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`the portion; (g) a primer extension preparation module for adding primer extension reaction
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`materials to the portion; (h) a mass spectrometry preparation module for removing a sample of
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`the portion from the second container to a support (e.g., chip or microwell) for analysis by mass
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`spectrometry; and (i) a mass spectrometry module for analyzing the sample.
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`The central controller can be a computer system, e.g., a commercially available personal
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`computer system, and can include linking software that enables the central controller to
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`communicate with at least one other module in the system. The system can also include a plate
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`editor module that provides sample information to the PCR preparation module, a transport
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`module comprising one or more robotic arms or tracks to transport a biological sample, or
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`portion thereof, between at least two modules of (a) to (i), and arranged to receive information
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`from and transmit information to the central controller. The system can also include a detection
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`module for detecting the presence of a sample and monitoring the progress of the sample through
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`the system, and arranged to receive information from and transmit information to the central
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`controller. The nucleic acids measurement system can include an ultraviolet light
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`spectrophotometer or a fluorometer. The PCR preparation module can include a pip etting robot,
`and the thermocycling system can include a thermocyler. The system can further include a
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`computer—readable medium comprising one or more programs for instructing a given module.
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`The PCR preparation module can include PCR materials, e. g., at least one primer set
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`described herein, e.g., a primer set selected from among SEQ ID NOS:1 to 504, each primer set
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`including two amplification primers and one detection extension primer. The sample transfer
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`system can include a pipetting robot.
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`In another aspect, the invention provides a method of performing a diagnostic assay on a
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`biological sample. The method includes (a) performing on a biological sample an assay using a
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`clinical assay system, wherein the assay comprises mass spectrometry analysis of a target nucleic
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`acid; and (b) automatically reporting information about the assay from a central controller of the
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`clinical assay system to an outside system or database accessible by at least one health care
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`provider (e.g., at least 2, 10, or more than 10) or at least one third party payor (e. g., at least 2, 10,
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`or more than 10). The clinical assay system can include at least one component selected from the
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`group consisting of: a central controller, a sample transfer module, a nucleic acid extraction
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`module, a nucleic acid measurement module, a PCR preparation module, a thermocyling module,
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`a primer extension preparation module, a mass spectrometry preparation module, and a mass
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`spectrometry module.
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`In another aspect, the invention provides a method of performing a diagnostic assay on a
`
`biological sample. The method includes (a) receiving a biological sample, generating
`
`information about the biological sample, and transmitting the information to a central controller;
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`(b) transferring a portion of the biological sample to a first container; (0) extracting nucleic acids
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`from cells within the portion and transferring the portion to a second container; ((1) measuring the
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`concentration of extracted nucleic acids in the portion; (6) adding polymerase chain reaction
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`(PCR) materials to the portion; (f) amplifying target nucleic acids in the portion; (g) adding
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`primer extension reaction materials to the portion; (11) extending a detection extension primer in
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`the portion; (i) transferring a sample of the portion from the second container to a supp01t;
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`(j) analyzing the sample and exporting data to the central controller using a mass spectrometry
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`system; and (k) transmitting the data from the central controller to an output device, external
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`system, or database. In certain embodiments, steps (a) to (k) can be performed automatically by
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`an automated system. The automated system can include at least one component selected from
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`the group consisting of: a central controller, a sample transfer module, a nucleic acid extraction
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`module, a nucleic acid measurement module, a PCR preparation module, a thermocyling module,
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`a primer extension preparation module, a mass spectrometry preparation module, and a mass
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`spectrometry module.
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`In certain embodiments, the diagnostic assay can be an assay for detecting mutations in a
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`gene. The gene can be a gene selected from the group consisting of: 5,10—
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`Methylenetetrahydrofolate Reductase (MTFR); Coagulation Factor II; Coagulation Factor V;
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`hemochromatosis (HFE); and a glucocerebrosidase (GC). fibroblast growth factor receptor 3;
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`aspartoacylase; Glucocerebrosidase; Coagulation Factor VII; Fanconi Anemia, Complementation
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`Group C (FANCC); inhibitor of kappa light polypeptide gene enhancer in b cells, kinase
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`complex-associated protein; acid sphingomyelinase; hexosaminidase; angiotensin i—converting
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`enzyme; adenylate cyclase 9; apolipoprotein A—l; apolipoprotein E; endothelial leukocyte
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`adhesion molecule 1; fc fragment of IGG, low affinity Ha, receptor; fibrinogen beta
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`chain;coagulation factor II, factor XIII; guanine nucleotide—binding protein beta—3; integrin,
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`alpha-2, glycoprotein Ia/Iia; glycoprotein Ib, platelet, alpha polypeptide; intercellular adhesion
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`molecule 1; glycoprotein Ia/IIa (a2), integrin, alpha—2; platelet glycoprotein Iib, integrin, alpha-
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`2b; glycoprotein IIb/IIIa, integrin, beta-3;3~hydroxy—3—methylglutaryl-coa reductase; lymphocyte
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`adhesion molecule 1; methylene tetrahydrofolate reductase; plasminogen activator inhibitor 1;
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`platelet alpha—granule membrane protein; transforming growth factor—beta receptor, type III;
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`thrombomodulin; tumor necrosis factor; vascular cell adhesion molecule; coagulation factor II
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`receptor; glycoprotein VI, platelet; purinergic receptor P2Y,g protein—coupled, 1; purinergic
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`receptor P2Y, G protein-coupled, 12; prostaglandin-endoperoxide synthase 1; prostaglandin—
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`endoperoxide synthase 2; thromboxane A2 receptor, platelet; and thrombospondin I.
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`In other embodiments, the diagnostic assay is an assay for detecting a pathogen in the
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`sample, e. g., a Virus, bacterium, or fungus. The virus can be a virus of the family Herpesviridae,
`
`e. g., cytomegalovirus (CMV).
`
`In another aspect, the invention provides an method, e.g., an automated method, for
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`detecting mutations in a target gene. The method includes a) amplifying a target sequence using
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`PCR and performing, e. g., automatically, a primer extension reaction using a set of three primers,
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`each set of primers including two amplification primers and one detection extension primer; b)r
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`transferring, e. g., automatically, detection extension primers to a mass spectrometry device; and
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`c) determining, e. g., automatically, the molecular weights of the detection extension primers by
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`mass spectrometry following the primer extension reaction, wherein a change in the molecular
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`weight of the extended primer, as compared to a control, indicates the presence of a mutation in
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`the gene. The method can include automatically transmitting information related to the presence
`
`of the mutation to a central controller.
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`In certain embodiments, the gene is a 5,10~Methylenetetrahydrofolate Reductase (MTFR)
`
`gene, and the set of three primers is selected from the group .consisting of: SEQ ID NOS: 1, 2,
`
`and 3; SEQ ID NOS: 4, 5 and 6; SEQ ID NOS: 7, 8, and 9; and SEQ ID NOS: 10,11, and 12;
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`each set of primers including two amplification primers and one detection extension primer.
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`In other embodiments, the gene is a Coagulation Factor II gene, and the set of three
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`primers is selected fiom the group consisting of: SEQ lD NOS: 13, 14, and 15 and SEQ ID NOS:
`
`16, 17 and 18; each primer set including two amplification primers and one detection extension
`
`primer.
`
`In still other embodiments, the gene is a Coagulation Factor Vgene, and the set of three
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`primers is selected from the group consisting of: SEQ ID NOS: 19, 20, and 21 or SEQ ID NOS:
`
`22, 23 and 24; each primer set including two amplification primers and one detection extension
`
`primer.
`
`In yet other embodiments, the gene is a hemochromatosis (HFE) gene, and the set of
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`three primers is selected from the group consisting of: SEQ ID NOS: 40, 41, and 42, SEQ ID
`
`NOS: 43, 44 and 45; SEQ ID NOS: 46, 47 and 48; SEQ 1D NOS: 49, 50 and 51; SEQ ID NOS:
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`52, 53 and 54; or SEQ ID NOS: 55, 56 and 57; each set of primers including two amplification
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`primers and one detection extension primer.
`
`In another aspect, the invention includes a method, e.g., an automated method, for
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`detecting a pathogen in a biological sample. The method includes a) amplifying a target
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`sequence using PCR and performing, e.g., automatically, a primer extension reaction using a set
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`of three primers, each set of primers including two amplification primers and one detection
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`extension primer; b) transferring,e.g., automatically, detection extension primers to a mass
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`spectrometry device; and c) determining, e.g., automatically, the molecular weights of the
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`detection extension primers by mass spectrometry following the primer extension reaction,
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`wherein a change in the molecular weight of the extended primer, as compared to controls,
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`indicates the presence presence of a pathogen in the sample. The controls can include an internal
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`control for determining the amount of the pathogen in the sample.
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`In some embodiments, the the pathogen is cytomegalovirus (CMV), and the three primers
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`are selected from the group consisting of: SEQ ID NOS: 25, 26, and 27; SEQ ID NOS: 28, 29
`
`and 30; SEQ ID NOS: 31, 32, and 33; SEQ ID NOS: 34, 35, and 36; SEQ ID NOS: 37, 38, and
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`39; and SEQ ID NOS: 58, 59, and 60; each primer set including two amplification primers and
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`one detection extension primer.
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`In another aspect, the invention includes an isolated DNA selected from the group
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`consisting of SEQ ID NOS.:1 to 504.
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`In still another aspect, the invention includes a kit that includes at least one primer set
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`described herein, e. g., a primer set selected from among SEQ ID NOS:1 to 504, each primer set
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`including two amplification primers and one detection extension primer, and instructions for
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`using the primer set to detect or analyze a target nucleic acid sequence in a biological sample.
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`For example, instructions can be provided to describe how to use the primers to detect the
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`presence of, or identify mutations in, a paiticular nucleic acid sequence or gene. As another
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`example, the instructions can describe how to use the primers to detect the presence of a
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`pathogen (e.g., a virus, bacterium, and/or fungus), the quantity of the pathogen, and/or the
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`genotype of the pathogen.
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`In yet another aspect, the invention includes a computer readable medium that includes a
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`program for instructing a central controller in an automated system for performing an assay on a
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`biological sample to: (a) receive a biological sample, generate information about the biological
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`sample, and transmit the information into a central controller; (b) transfer a portion of the
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`biological sample to a first container;
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`(0) extract nucleic acids from cells within the portion and
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`transfer the portion to a second container; (d) measure the concentration of extracted nucleic
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`acids in the portion; (e) add polymerase chain reaction (PCR) materials to the portion; (t)
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`amplify target nucleic acids in the portion; (g) add primer extension reaction materials to the
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`portion; (h) extend a detection extension primer in the portion; (i) transfer a sample of the portion
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`from the second container to a support; (j) analyze the sample and exporting data to the central
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`controller using a mass spectrometry system; and (k) transmit the data from the central controller
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`to an output device, external system, or database.
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`Unless otherwise defined, all technical and scientific terms used herein have the same
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`meaning as commonly understood by one of ordinary skill in the art to which this invention
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`belongs. Although methods and equipment or software similar or equivalent to those described
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`herein can be used in the practice of the present invention, suitable methods, equipment, and
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`software are described below. All publications and other references mentioned herein are
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`incorporated by reference in their entirety. In case of conflict, the present specification,
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`including definitions, will control. In addition, the materials, methods, and examples are
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`illustrative only and not intended to be limiting.
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`The details of one or more embodiments of the invention are set forth in the accompa-
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`nying drawings and the description below. Other features and advantages of the invention will
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`be apparent from the description and drawings, and from the claims.
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`DESCRIPTION OF DRAWINGS
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`FIG. 1 is a diagram illustrating the main components of a clinical assay system and the
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`flow of biological samples and information through the system.
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`FIG. 2 is a flow diagram illustrating the steps of the clinical assay system.
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`FIG. 3 is a mass spectrum of a heterozygous “TC” allele (heterozygous positive)
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`generated using a screen for a C677T mutation in the 5,10—Methylenetetrahydrofolate Reductase
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`(MTFR) gene.
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`FIG. 4 is a mass spectrum of a heterozygous “GA” allele (heterozygous positive)
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`generated using a screen for a G20210A mutation in the Coagulation Factor II (FII) gene.
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`FIG. 5 is a mass spectrum of a heterozygous “GA” allele (heterozygous positive)
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`generated using a screen for a R506Q mutation in the Coagulation Factor V gene.
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`FIG. 6 is a mass spectrum of a heterozygous “GA” allele (heterozygous positive)
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`generated using a screen for a R506Q mutation in the Coagulation Factor V gene.
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`FIG. 7 is a mass spectrum of heterozygous “GC” alleles (heterozygous positive) for
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`H63D Histidine to Aspartic acid (C187G) mutation in the FM3 —E assay.
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`FIG. 8 is a mass spectrum of heterozygous “GC” alleles (heterozygous positive) for
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`H63D Histidine to Aspartic acid (C187G) mutation in the HFE-E3 assay.
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`FIG. 9 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for S65C
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`Serine to Cysteine (A193 T) mutation in the HFE SGSC El assay.
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`FIG. 10 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for
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`S65 C Serine to Cysteine (A193T) mutation in the HFE S65C E5 assay.
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`FIG. 11 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for
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`C282Y cysteine to tyrosine (G845A) mutation in the FM6-E assay.
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`FIG. 12 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for
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`C282Y cysteine to tyrosine (G845A) mutation in the HFE-E6 assay.
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`FIG. l3Al~13A3 is a set of mass spectra in a CMV quantitative assay on samples
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`containing 400 CMV copies/ml.
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`FIG. 13B1-13B3 is a set of mass spectra in a CMV quantitative assay on samples
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`containing 4000 CMV copies/ml.
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`FIG. 13C1-13C3 is a set of mass spectra in a CMV quantitative assay on samples
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`containing 40,000 CMV copies/ml.
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`FIG. 13D—13D3 is a set of mass spectra in a CMV quantitative assay on samples
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`containing 400,000 CMV copies/ml.
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`FIG. 13E1—13E3 is a set of mass spectra in a CMV quantitative assay on samples
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`containing 4,000,000 CMV copies/ml.
`
`FIG. 13F1-13F3 is a set of mass spectra in a CMV quantitative assay on samples
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`containing 40,000,000 CMV copies/m1.
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`FIG. 13G1-13G3 is a set of mass spectra in a CMV quantitative assay on samples
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`containing 400,000,000 CMV copies/ml.
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`FIG. 14 is a graph that plots CMV plasma samples versus internal standards. A CMV
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`control (4x109 copies per ml) was diluted down to 40 copies/ml in 10—fold increments, mixed
`with the Internal Standard of appropriate concentration, extracted (240 pl) on IWDX (Qiagen),
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`eluted in 75 ul of buffer and assayed (2 ul) by PCR, followed by SAP treatment, extension
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`reaction and mass spectrometry analysis.
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`FIG. 15A-15GG is a table that lists a number of genetic targets for the assays of the
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`invention, along with exemplary primers for those targets.
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`DETAILED DESCRIPTION
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`The invention provides a new highly automated system for performing clinical assays,
`optionally with automatic billing to third party providers such as insurance companies. The
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`invention also provides novel assays using mass spectrometry (e.g., matrix—assisted laser
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`desorption/ionization (lVIALDI). The assays are highly accurate and can detect, for example,
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`sequence variations (e.g., mutations and/or polymorphisms) and foreign sequences (e. g., viral
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`sequences) incorporated into a target gene. The assays are also useful for infectious
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`disease/patho gen testing.
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`The entire process, or portions thereof, can be automated, i.e., performed by machine(s).
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`Accordingly, the present invention also includes a high—throughput process for performing the
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`assays described herein. Thus, the new system can perform dozens (e. g., 96, 128, 384) of
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`different assays on dozens of different biological samples at the same time.
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`Clinical Assay System
`
`Overview ofSystem
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`FIG. 1 provides an overview of the clinical assay system 2 of the present invention. The
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`clinical assay system includes, as its main components, the following modules. A central
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`controller 4 for exchanging information about the biological sample with an outside system or
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`database 8 and with one or more modules or systems within clinical assay system, and an input
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`device 6; a sample transfer system 10 for transferring a portion of the sample to a first container;
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`a nucleic acid extraction system 12 for extracting nucleic acids from the portion and for
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`transferring the portion from the first container to a second container; a nucleic acid
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`measurement system 14 for measuring the concentration of nucleic acids in the portion; a PCR
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`preparation system 16 for adding polymerase chain reaction (PCR) reaction materials to the
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`portion; a thermocycling system 18 for amplifying a target sequence and extending a primer in
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`the portion; a primer extension preparation system 20 for adding primer extension reaction
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`materials to the portion; a mass spectrometry preparation system 22 for removing a sample of the
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`portion from the second container to a platform for analysis by mass spectrometry; and a mass
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`spectrometry system 24 for analyzing the sample.
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`The central controller is capable of controlling one or more system modules, collecting
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`and organizing data obtained from one or more of the system modules and an outside system or
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`database, and of sending data to one or more of the system modules and an outside database
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`(e.g., a database accessible by healthcare providers or third parties) or system (e.g., an outside
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`computer through which health care providers or third parties can access the data). The input
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`device 6 associated with the central controller can be a bar code reader. The system can
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`optionally include a detection system 5 for detecting and tracking a sample as it progresses
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`through the system. The system can also include a transport subsystem 25, e.g., a system of one
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`or more robotic arms and/or tracks, for transporting samples between two or more modules
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`within the system.
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`Central Controller
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`Typically, the central controller 4 is a computer system. The computer systems that can
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`be used are commonly available personal computers having read-write memory, or industrial
`
`counterparts thereof. The central controller is provided with a suitable input device 6 such as a
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`keyboard, touch screen, card reader, bar code scanner, or another computer (e.g., for inputting
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`biological sample processing instructions and patient identification information).
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`The central controller 4 is run by linking software, which directs the central controller to
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`receive information from, and/or transmit information to, each of the modules in the overall
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`system. For example, the central controller can be configured to exchange information with one
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`or more modules within the clinical assay system, and to relay that information to one or more
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`other modules. Such information may include information about a biological sample, e. g.,
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`sample identification, information as to which assay(s) is to be/has been performed on a sample,
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`and the location of a sample within the clinical assay system and within/a given batch of samples
`being processed.
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`The central controller 4 is also configured to exchange information with outside systems
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`and/or databases 8 (i.e., systems or databases not part of the clinical assay system). This
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`configuration allows the central controller to report, e.g., the results of the clinical assays
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`described herein, along with other data, e. g., billing amounts, patient identification, and other
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`data to health—care providers (e.g., technicians, nurses, physicians) and/or third parties (e. g.,
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`insurance providers) at other sites. Reporting can occur automatically. Exemplary of outside
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`systems are systems capable of interfacing directly with the central controller, or with a database
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`accessible by both the outside system and the central controller. For example, MeditechTM
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`provides a laboratory application that allows multisite and/or multifacility specimen tracking,
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`through which the central controller can exchange information with outside systems.
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`Sample Transfer Module
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`The sample transfer system 10 can be any system capable of receiving a biological
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`sample, e. g., a blood sample, removing an aliquot of the sample, and placing the aliquot into one
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`or more receptacles. Exemplary systems are pipetting robots, such as the Genesis® FreedomTM
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`Automated Workstation. The system is capable of scanning sample tube barcodes and multiwell
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`(e.g., 96-well) plates, and creating a file that indicates where on the multiwell plate a sample is
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`located following the‘ transfer. The file can include information such as the barcodes of scanned
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`sample tubes, the location of these samples on the multiwell plate, the volume transferred from
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`the sample tube to the plate, and overall identifying information (e. g., a barcode) for of the
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`multiwell plate (called DNA plate).
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`Nucleic Acid Extraction Module
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`The nucleic acid extraction system 12 can be any system capable of carrying out
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`techniques, such as those described herein, for purifying nucleic acids (i.e., DNA and/or RNA)
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`from one or more biological samples. An example of such a system is the BioRobot® MDx
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`produced by Qiagen.
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`Nucleic Acids Measurement Module
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`The nucleic acids measurement system 14 can be any system capable of measuring the
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`concentration of nucleic acids in a sample. For example, the system can be a commercially
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`available ultraviolet (UV) light spectrophotometer, which is capable of determining the
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`concentration of nucleic acids using optical density measurements. As another example, the
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`system can be capable of measuring the UV-induced fluorescence of dye (e.g., ethidium bromide
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`or Pico Green) intercalated into the nucleic acid, such as a fluorometer. The Genesis®
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`FreedomTM Automated Workstation produced by Tecan can include such a fluorometer. The
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`nucleic acid measurement system can be associated with (e. g., a part of) the sample transfer
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`system, or it can be a stand-alone module.
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`PCR Preparation Module
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`The PCR preparation system 16 can be any system capable of adding appropriate
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`materials, e. g., enzymes (e. g., Taq polymerase), nucleic acid primers, individual nucleotides, and
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`reagents, to an aliquot in preparation for amplifying a target sequence in the aliquot. The PCR
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`preparation also prepares appropriate control reaction mixes. Examples of such systems are the
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`Genesis® Automated Workstation and the Tecan TeMOTM multi—pipetting module.
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`Overall, the PCR preparation system is capable of performing at least two steps. The first
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`is to dispense appropriate assay mixes. Assay mixes can be prepared by an individual, e. g., a
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`technician, or by a robot, according to typical laboratory procedures, and placed into holders.
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`These PCR preparation system dispenses the mixes from the holders to a position on a second
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`multiwell (e.g., 384) plate, according to instructions (e.g., sample identification and assays to be
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`performed) it receives from a plate editor 7 (described in detail below). The second is to add
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`samples to the appropriate assay mix. Using the file received by the PCR preparation system 16
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`from the sample transfer system 10, the PCR preparation system transfers samples from the first
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`multiwell (e.g., 96 well) plate to the second multiwell (e.g., 384 well) plate. In this way, the
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`PCR preparation system is able to transfer samples from a first plate to a second plate, while
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`keeping track of the location of the samples, and to ensure that the appropriate assays are
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`performed on each sample.
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`Thermocycling Module
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`The thermocycling system 18 can be any‘ system capable of performing PCR reactions,
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`e. g., PCR amplification and/or primer extension reactions, and is typically a commercially
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`available thermocycler. Exemplary systems include the GeneAmp PCR System 9700
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`manufactured by Applied Biosystems, the Perkin Elmer 2000 PCR thennocycler, and the PTC—
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`200 thennocycler manufactured by MJ Research.
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`Primer Extension Preparation Module
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`The primer extension preparation system 20 can be any system capable of adding
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`appropriate materials, e. g., Shrimp Alkaline Phosphatase (SAP; to dephosphorylate
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`unincorporated dNTPs), extension primers (e.g., the extension primers described herein), and
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`appropriate mixtures of dNTPs and ddNTPs, to an aliquot in preparation for performing primer
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`extension reactions. An exemplary system is the MultimekTM manufactured by Beckman—
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`Coulter
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`Mass Spectrometry Preparation Module
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`The mass spectrometry preparation system 22 can be any system capable of removing a
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`sample of an aliquot and placing the sample on a support, e.g., a chip, for analysis by mass
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`spectroscopy. The support can be composed of any material known to those skilled in the art to
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`be usable in mass spectrometry, e.g., silicon, plastic, glass, and/or ceramic. A Wide variety of
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`chips are commercially available. Exemplary of chips is the Sequenom® SpectroCHIPTM, which
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`is supplied in 384 well format and are pre—spotted with a specially formulated matrix assisted
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`laser desorption ionization (MALDI) matrix. The matrix can be of any composition known in
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`the art of mass spectrometry, e. g., 0! —cyano-4-hydroxy cinnamic acid (CHCA), 2,4,6-trihydroxy
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`acetophenone (THAP), or 3—dydroxypicolinic acid (3—HPA) in ammonium citrate, the choice of
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`which will depend, e.g., on the mass spectrometry system used and the assay to be perform