# Patent

## Title
Systems and methods for analyzing nucleic acid sequences

## Abstract

The invention relates to systems and methods for analyzing clinically relevant nucleic acid sequences.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application Nos. 60/493,238, filed on Aug. 6, 2003, and 60/568,958, filed on May 7, 2004. The contents of both of those provisional applications is incorporated herein by reference in its entirety.


TECHNICAL FIELD
This invention relates to systems and methods for analyzing clinically relevant nucleic acid sequences.
BACKGROUND
The healthcare delivery system has changed remarkably over the past several decades. Clinical laboratories are under increasing pressure to deliver low cost and highly accurate analytical services with the rapid turn-around time required by physicians and patients. Laboratory testing has changed and improved in recent years to meet the challenge. Robotics has been introduced to the laboratory to increase efficiency and reduce the need for human participation, and laboratory instruments have been designed to decrease the biological sample volumes needed to perform various assays. However, more improvement in the clinical laboratory area is required to meet the demands of the ever-changing healthcare system.
SUMMARY
The present invention provides novel automated systems and methods to perform assays on nucleic acid sequences (e.g., clinically relevant nucleic acid sequences). The system can provide assay results quickly, accurately, and in a format easily accessible by health care providers and/or third party payors (e.g., insurance companies). The invention also provides novel and highly accurate assays using mass spectrometry (e.g., matrix-assisted laser desorption/ionization (MALDI)).
In one aspect, the invention provides a system for performing a diagnostic assay on a biological sample. The system includes, as its main components (a) a central controller programmed to: (i) exchange information about the biological sample with an outside system or database; and (ii) exchange information about the biological sample with one or more modules of the system; (b) a sample transfer module for transferring a portion of the sample to a first container; (c) a nucleic acid extraction module for extracting nucleic acids from cells within the portion and for transferring the portion from the first container to a second container; (d) a nucleic acid measurement module for measuring the concentration of nucleic acids in the portion; (e) a PCR preparation module for adding polymerase chain reaction (PCR) reaction materials (e.g., individual nucleotides, primers, polymerase enzymes, and reagents) to the portion; (f) a thermocyling module for amplifying a target sequence and extending a primer in the portion; (g) a primer extension preparation module for adding primer extension reaction materials to the portion; (h) a mass spectrometry preparation module for removing a sample of the portion from the second container to a support (e.g., chip or microwell) for analysis by mass spectrometry; and (i) a mass spectrometry module for analyzing the sample.
The central controller can be a computer system, e.g., a commercially available personal computer system, and can include linking software that enables the central controller to communicate with at least one other module in the system. The system can also include a plate editor module that provides sample information to the PCR preparation module, a transport module comprising one or more robotic arms or tracks to transport a biological sample, or portion thereof, between at least two modules of (a) to (i), and arranged to receive information from and transmit information to the central controller. The system can also include a detection module for detecting the presence of a sample and monitoring the progress of the sample through the system, and arranged to receive information from and transmit information to the central controller. The nucleic acids measurement system can include an ultraviolet light spectrophotometer or a fluorometer. The PCR preparation module can include a pipetting robot, and the thermocycling system can include a thermocyler. The system can further include a computer-readable medium comprising one or more programs for instructing a given module.
The PCR preparation module can include PCR materials, e.g., at least one primer set described herein, e.g., a primer set selected from among SEQ ID NOS:1 to 504, each primer set including two amplification primers and one detection extension primer. The sample transfer system can include a pipetting robot.
In another aspect, the invention provides a method of performing a diagnostic assay on a biological sample. The method includes (a) performing on a biological sample an assay using a clinical assay system; wherein the assay comprises mass spectrometry analysis of a target nucleic acid; and (b) automatically reporting information about the assay from a central controller of the clinical assay system to an outside system or database accessible by at least one health care provider (e.g., at least 2, 10, or more than 10) or at least one third party payor (e.g., at least 2, 10, or more than 10). The clinical assay system can include at least one component selected from the group consisting of: a central controller, a sample transfer module, a nucleic acid extraction module, a nucleic acid measurement module, a PCR preparation module, a thermocyling module, a primer extension preparation module, a mass spectrometry preparation module, and a mass spectrometry module.
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; (b) transferring a portion of the biological sample to a first container; (c) extracting nucleic acids from cells within the portion and transferring the portion to a second container; (d) measuring the concentration of extracted nucleic acids in the portion; (e) adding polymerase chain reaction (PCR) materials to the portion; (f) amplifying target nucleic acids in the portion; (g) adding primer extension reaction materials to the portion; (h) extending a detection extension primer in the portion; (i) transferring a sample of the portion from the second container to a support; (j) analyzing the sample and exporting data to the central controller using a mass spectrometry system; and (k) transmitting the data from the central controller to an output device, external system, or database. In certain embodiments, steps (a) to (k) can be performed automatically by an automated system. The automated system can include at least one component selected from the group consisting of: a central controller, a sample transfer module, a nucleic acid extraction module, a nucleic acid measurement module, a PCR preparation module, a thermocyling module, a primer extension preparation module, a mass spectrometry preparation module, and a mass spectrometry module.
In certain embodiments, the diagnostic assay can be an assay for detecting mutations in a gene. The gene can be a gene selected from the group consisting of: 5,10-Methylenetetrahydrofolate Reductase (MTFR); Coagulation Factor II; Coagulation Factor V; hemochromatosis (IFE); and a glucocerebrosidase (GC). fibroblast growth factor receptor 3; aspartoacylase; Glucocerebrosidase; Coagulation Factor VII; Fanconi Anemia, Complementation Group C (FANCC); inhibitor of kappa light polypeptide gene enhancer in b cells, kinase complex-associated protein; acid sphingomyelinase; hexosaminidase; angiotensin i-converting enzyme; adenylate cyclase 9; apolipoprotein A-1; apolipoprotein E; endothelial leukocyte adhesion molecule 1; fc fragment of IGG, low affinity IIIa, receptor; fibrinogen beta chain; coagulation factor II, factor XIII; guanine nucleotide-binding protein beta-3; integrin, alpha-2, glycoprotein Ia/IIa; glycoprotein Ib, platelet, alpha polypeptide; intercellular adhesion molecule 1; glycoprotein Ia/IIa (a2), integrin, alpha-2; platelet glycoprotein Iib, integrin, alpha-2b; glycoprotein IIb/IIIa, integrin, beta-3;3-hydroxy-3-methylglutaryl-coa reductase; lymphocyte adhesion molecule 1; methylene tetrahydrofolate reductase; plasminogen activator inhibitor 1; platelet alpha-granule membrane protein; transforming growth factor-beta receptor, type III; thrombomodulin; tumor necrosis factor; vascular cell adhesion molecule; coagulation factor II receptor; glycoprotein VI, platelet; purinergic receptor P2Y, g protein-coupled, 1; purinergic receptor P2Y, G protein-coupled, 12; prostaglandin-endoperoxide synthase 1; prostaglandin-endoperoxide synthase 2; thromboxane A2 receptor, platelet; and thrombospondin I.
In other embodiments, the diagnostic assay is an assay for detecting a pathogen in the 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 detecting mutations in a target gene. The method includes a) amplifying a target sequence using PCR and performing, e.g., automatically, a primer extension reaction using a set of three primers, each set of primers including two amplification primers and one detection extension primer; b) transferring, e.g., automatically, detection extension primers to a mass spectrometry device; and c) determining, e.g., automatically, the molecular weights of the detection extension primers by mass spectrometry following the primer extension reaction, wherein a change in the molecular weight of the extended primer, as compared to a control, indicates the presence of a mutation in the gene. The method can include automatically transmitting information related to the presence of the mutation to a central controller.
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; each set of primers including two amplification primers and one detection extension primer.
In other embodiments, the gene is a Coagulation Factor II gene, and the set of three primers is selected from the group consisting of: SEQ ID 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 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 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 ID NOS: 49, 50 and 51; SEQ ID NOS: 52, 53 and 54; or SEQ ID NOS: 55, 56 and 57; each set of primers including two amplification primers and one detection extension primer.
In another aspect, the invention includes a method, e.g., an automated method, for detecting a pathogen in a biological sample. The method includes a) amplifying a target sequence using PCR and performing, e.g., automatically, a primer extension reaction using a set of three primers, each set of primers including two amplification primers and one detection extension primer; b) transferring, e.g., automatically, detection extension primers to a mass spectrometry device; and c) determining, e.g., automatically, the molecular weights of the detection extension primers by mass spectrometry following the primer extension reaction, wherein a change in the molecular weight of the extended primer, as compared to controls, indicates the presence of a pathogen in the sample. The controls can include an internal control for determining the amount of the pathogen in the sample.
In some embodiments, the pathogen is cytomegalovirus (CMV), and the three primers 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 39; and SEQ ID NOS: 58, 59, and 60; each primer set including two amplification primers and one detection extension primer.
In another aspect, the invention includes an isolated DNA selected from the group consisting of SEQ ID NOS.:1 to 504.
In still another aspect, the invention includes a kit that includes at least one primer set described herein, e.g., a primer set selected from among SEQ ID NOS: 1 to 504, each primer set including two amplification primers and one detection extension primer, and instructions for using the primer set to detect or analyze a target nucleic acid sequence in a biological sample. For example, instructions can be provided to describe how to use the primers to detect the presence of, or identify mutations in, a particular nucleic acid sequence or gene. As another example, the instructions can describe how to use the primers to detect the presence of a pathogen (e.g., a virus, bacterium, and/or fungus), the quantity of the pathogen, and/or the genotype of the pathogen.
In yet another aspect, the invention includes a computer readable medium that includes a program for instructing a central controller in an automated system for performing an assay on a biological sample to: (a) receive a biological sample, generate information about the biological sample, and transmit the information into a central controller; (b) transfer a portion of the biological sample to a first container; (c) extract nucleic acids from cells within the portion and transfer the portion to a second container; (d) measure the concentration of extracted nucleic acids in the portion; (e) add polymerase chain reaction (PCR) materials to the portion; (f) amplify target nucleic acids in the portion; (g) add primer extension reaction materials to the portion; (h) extend a detection extension primer in the portion; (i) transfer a sample of the portion from the second container to a support; (j) analyze the sample and exporting data to the central controller using a mass spectrometry system; and (k) transmit the data from the central controller to an output device, external system, or database.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and equipment or software similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods, equipment, and software are described below. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the description and drawings, and from the claims.



DESCRIPTION OF DRAWINGS
FIG. 1 is a diagram illustrating the main components of a clinical assay system and the flow of biological samples and information through the system.
FIG. 2 is a flow diagram illustrating the steps of the clinical assay system.
FIG. 3 is a mass spectrum of a heterozygous “TC” allele (heterozygous positive) generated using a screen for a C677T mutation in the 5,10-Methylenetetrahydrofolate Reductase (MTFR) gene.
FIG. 4 is a mass spectrum of a heterozygous “GA” allele (heterozygous positive) generated using a screen for a G20210A mutation in the Coagulation Factor II (FII) gene.
FIG. 5 is a mass spectrum of a heterozygous “GA” allele (heterozygous positive) generated using a screen for a R506Q mutation in the Coagulation Factor V gene.
FIG. 6 is a mass spectrum of a heterozygous “GA” allele (heterozygous positive) generated using a screen for a R506Q mutation in the Coagulation Factor V gene.
FIG. 7 is a mass spectrum of heterozygous “GC” alleles (heterozygous positive) for H63D Histidine to Aspartic acid (C187G) mutation in the FM3-E assay.
FIG. 8 is a mass spectrum of heterozygous “GC” alleles (heterozygous positive) for H63D Histidine to Aspartic acid (C187G) mutation in the HFE-E3 assay.
FIG. 9 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for S65C Serine to Cysteine (A193T) mutation in the HFE S65C E1 assay.
FIG. 10 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for S65C Serine to Cysteine (A193T) mutation in the HFE S65C E5 assay.
FIG. 11 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for C282Y cysteine to tyrosine (G845A) mutation in the FM6-E assay.
FIG. 12 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for C282Y cysteine to tyrosine (G845A) mutation in the HFE-E6 assay.
FIGS. 13A1-13A3 is a set of mass spectra in a CMV quantitative assay on samples containing 400 CMV copies/ml.
FIGS. 13B1-13B3 is a set of mass spectra in a CMV quantitative assay on samples containing 4000 CMV copies/ml.
FIGS. 13C1-13C3 is a set of mass spectra in a CMV quantitative assay on samples containing 40,000 CMV copies/ml.
FIGS. 13D-13D3 is a set of mass spectra in a CMV quantitative assay on samples containing 400,000 CMV copies/ml.
FIGS. 13E1-13E3 is a set of mass spectra in a CMV quantitative assay on samples containing 4,000,000 CMV copies/ml.
FIGS. 13F1-13F3 is a set of mass spectra in a CMV quantitative assay on samples containing 40,000,000 CMV copies/ml.
FIGS. 13G1-13G3 is a set of mass spectra in a CMV quantitative assay on samples containing 400,000,000 CMV copies/ml.
FIG. 14 is a graph that plots CMV plasma samples versus internal standards. A CMV control (4×109 copies per ml) was diluted down to 40 copies/ml in 10-fold increments, mixed with the Internal Standard of appropriate concentration, extracted (240 μl) on MDX (Qiagen), eluted in 75 μl of buffer and assayed (2 μl) by PCR, followed by SAP treatment, extension reaction and mass spectrometry analysis.
FIGS. 15A-15GG is a table that lists a number of genetic targets for the assays of the invention, along with exemplary primers for those targets.



DETAILED DESCRIPTION
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 invention also provides novel assays using mass spectrometry (e.g., matrix-assisted laser desorption/ionization (MALDI). The assays are highly accurate and can detect, for example, sequence variations (e.g., mutations and/or polymorphisms) and foreign sequences (e.g., viral sequences) incorporated into a target gene. The assays are also useful for infectious disease/pathogen testing.
The entire process, or portions thereof, can be automated, i.e., performed by machine(s). Accordingly, the present invention also includes a high-throughput process for performing the assays described herein. Thus, the new system can perform dozens (e.g., 96, 128, 384) of different assays on dozens of different biological samples at the same time.
Clinical Assay System
Overview of System
FIG. 1 provides an overview of the clinical assay system 2 of the present invention. The clinical assay system includes, as its main components, the following modules. A central controller 4 for exchanging information about the biological sample with an outside system or database 8 and with one or more modules or systems within clinical assay system, and an input device 6; a sample transfer system 10 for transferring a portion of the sample to a first container; a nucleic acid extraction system 12 for extracting nucleic acids from the portion and for transferring the portion from the first container to a second container; a nucleic acid measurement system 14 for measuring the concentration of nucleic acids in the portion; a PCR preparation system 16 for adding polymerase chain reaction (PCR) reaction materials to the portion; a thermocycling system 18 for amplifying a target sequence and extending a primer in the portion; a primer extension preparation system 20 for adding primer extension reaction materials to the portion; a mass spectrometry preparation system 22 for removing a sample of the portion from the second container to a platform for analysis by mass spectrometry; and a mass spectrometry system 24 for analyzing the sample.
The central controller is capable of controlling one or more system modules, collecting and organizing data obtained from one or more of the system modules and an outside system or database, and of sending data to one or more of the system modules and an outside database (e.g., a database accessible by healthcare providers or third parties) or system (e.g., an outside computer through which health care providers or third parties can access the data). The input device 6 associated with the central controller can be a bar code reader. The system can optionally include a detection system 5 for detecting and tracking a sample as it progresses through the system. The system can also include a transport subsystem 25, e.g., a system of one or more robotic arms and/or tracks, for transporting samples between two or more modules within the system.
Central Controller
Typically, the central controller 4 is a computer system. The computer systems that can 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 keyboard, touch screen, card reader, bar code scanner, or another computer (e.g., for inputting biological sample processing instructions and patient identification information).
The central controller 4 is run by linking software, which directs the central controller to receive information from, and/or transmit information to, each of the modules in the overall system. For example, the central controller can be configured to exchange information with one or more modules within the clinical assay system, and to relay that information to one or more other modules. Such information may include information about a biological sample, e.g., sample identification, information as to which assay(s) is to be/has been performed on a sample, and the location of a sample within the clinical assay system and within a given batch of samples being processed.
The central controller 4 is also configured to exchange information with outside systems and/or databases 8 (i.e., systems or databases not part of the clinical assay system). This configuration allows the central controller to report, e.g., the results of the clinical assays described herein, along with other data, e.g., billing amounts, patient identification, and other data to health-care providers (e.g., technicians, nurses, physicians) and/or third parties (e.g., insurance providers) at other sites. Reporting can occur automatically. Exemplary of outside systems are systems capable of interfacing directly with the central controller, or with a database accessible by both the outside system and the central controller. For example, MeditechTM provides a laboratory application that allows multisite and/or multifacility specimen tracking, through which the central controller can exchange information with outside systems.
Sample Transfer Module
The sample transfer system 10 can be any system capable of receiving a biological sample, e.g., a blood sample, removing an aliquot of the sample, and placing the aliquot into one or more receptacles. Exemplary systems are pipetting robots, such as the Genesis® FreedomTM Automated Workstation. The system is capable of scanning sample tube barcodes and multiwell (e.g., 96-well) plates, and creating a file that indicates where on the multiwell plate a sample is located following the transfer. The file can include information such as the barcodes of scanned sample tubes, the location of these samples on the multiwell plate, the volume transferred from the sample tube to the plate, and overall identifying information (e.g., a barcode) for of the multiwell plate (called DNA plate).
Nucleic Acid Extraction Module
The nucleic acid extraction system 12 can be any system capable of carrying out techniques, such as those described herein, for purifying nucleic acids (i.e., DNA and/or RNA) from one or more biological samples. An example of such a system is the BioRobot® MDx produced by Qiagen.
Nucleic Acids Measurement Module
The nucleic acids measurement system 14 can be any system capable of measuring the concentration of nucleic acids in a sample. For example, the system can be a commercially available ultraviolet (UV) light spectrophotometer, which is capable of determining the concentration of nucleic acids using optical density measurements. As another example, the system can be capable of measuring the UV-induced fluorescence of dye (e.g., ethidium bromide or Pico Green) intercalated into the nucleic acid, such as a fluorometer. The Genesis® FreedomTM Automated Workstation produced by Tecan can include such a fluorometer. The nucleic acid measurement system can be associated with (e.g., a part of) the sample transfer system, or it can be a stand-alone module.
PCR Preparation Module
The PCR preparation system 16 can be any system capable of adding appropriate materials, e.g., enzymes (e.g., Taq polymerase), nucleic acid primers, individual nucleotides, and reagents, to an aliquot in preparation for amplifying a target sequence in the aliquot. The PCR preparation also prepares appropriate control reaction mixes. Examples of such systems are the Genesis® Automated Workstation and the Tecan TeMOTM multi-pipetting module.
Overall, the PCR preparation system is capable of performing at least two steps. The first is to dispense appropriate assay mixes. Assay mixes can be prepared by an individual, e.g., a technician, or by a robot, according to typical laboratory procedures, and placed into holders. These PCR preparation system dispenses the mixes from the holders to a position on a second multiwell (e.g., 384) plate, according to instructions (e.g., sample identification and assays to be performed) it receives from a plate editor 7 (described in detail below). The second is to add samples to the appropriate assay mix. Using the file received by the PCR preparation system 16 from the sample transfer system 10, the PCR preparation system transfers samples from the first multiwell (e.g., 96 well) plate to the second multiwell (e.g., 384 well) plate. In this way, the PCR preparation system is able to transfer samples from a first plate to a second plate, while keeping track of the location of the samples, and to ensure that the appropriate assays are performed on each sample.
Thermocycling Module
The thermocycling system 18 can be any system capable of performing PCR reactions, e.g., PCR amplification and/or primer extension reactions, and is typically a commercially available thermocycler. Exemplary systems include the GeneAmp PCR System 9700 manufactured by Applied Biosystems, the Perkin Elmer 2000 PCR thermocycler, and the PTC-200 thermocycler manufactured by MJ Research.
Primer Extension Preparation Module
The primer extension preparation system 20 can be any system capable of adding appropriate materials, e.g., Shrimp Alkaline Phosphatase (SAP; to dephosphorylate unincorporated dNTPs), extension primers (e.g., the extension primers described herein), and appropriate mixtures of dNTPs and ddNTPs, to an aliquot in preparation for performing primer extension reactions. An exemplary system is the MultimekTM manufactured by Beckman-Coulter
Mass Spectrometry Preparation Module
The mass spectrometry preparation system 22 can be any system capable of removing a sample of an aliquot and placing the sample on a support, e.g., a chip, for analysis by mass spectroscopy. The support can be composed of any material known to those skilled in the art to be usable in mass spectrometry, e.g., silicon, plastic, glass, and/or ceramic. A wide variety of chips are commercially available. Exemplary of chips is the Sequenom® SpectroCHIPTM, which is supplied in 384 well format and are pre-spotted with a specially formulated matrix assisted laser desorption ionization (MALDI) matrix. The matrix can be of any composition known in the art of mass spectrometry, e.g., α-cyano-4-hydroxy cinnamic acid (CHCA), 2,4,6-trihydroxy acetophenone (THAP), or 3-dydroxypicolinic acid (3-HPA) in ammonium citrate, the choice of which will depend, e.g., on the mass spectrometry system used and the assay to be performed. Exemplary of the mass spectrometry preparation systems is the Sequenom® SpectroPOINTTM, a nanoliter sample dispensing instrument.
Mass Spectrometry Module
The mass spectrometry system 24 can be any commercially available mass spectrometer. The clinical assay system of the present invention can be configured to utilize mass spectrometer formats including matrix assisted laser desorption ionization (MALDI), electrospray (ES), ion cyclotron resonance (ICR) and Fourier Transform. In one embodiment, maMALDI is utilized in the clinical assay system of the present invention. An exemplary mass spectrometry system is the Sequenom® AutoflexTM Mass Spectrometer.
With MALDI mass spectrometry, various mass analyzers can be used, e.g., magnetic sector/magnetic deflection instruments in single or triple quadrupole mode (MS/MS), Fourier transform and time-of-flight (TOF) configurations as is known in the art of mass spectrometry. For the desorption/ionization process, numerous matrix/laser combinations can be used. Ion-trap and reflectron configurations can also be employed. In one embodiment of the present invention, MALDI-TOF is employed to analyze the biological samples.
Transport Subsystem
Optionally, the clinical assay system includes a transport system 25. The transport system can be capable of (i) transporting containers, e.g., containing biological samples, from a source (e.g., a separate site in which biological samples are obtained from a patient) to the clinical assay system; (ii) transporting containers between at least two modules within the clinical assay system; and/or (iii) transporting containers away from the clinical assay system to a predefined destination after completion of the assay. The transport system is capable of communicating with the central controller so the central controller can direct the transport system and/or receive information as to the location of sample within the clinical assay system. The transport system can comprise one or more robotic arms or tracks, or a combination of robotic arms and tracks.
Detection System
Optionally, the clinical assay system includes a detection system 5 for detecting the presence of a biological sample and monitoring the progress of the sample through the system. The detection system is capable of receiving information from and transmitting information to the central controller. The detection system can include a network of sensors, e.g., barcode readers, reed switches, weight systems (where the detector detects the presence of a sample by its weight); or optical interrupters, or combinations thereof, arranged throughout the clinical assay system, each of which are capable of transmitting information, directly or indirectly, to the central controller.
Software
The clinical assay system includes novel software for adapting one or more modules to be used in the system. For example, the central controller includes a novel software program to provide an interface between itself and an outside system or database (e.g., Meditech). In addition to causing the central controller to receive data from an outside source, the software causes the central controller to send information (e.g., automatically) about the clinical assays (e.g., the results and/or billing information) to at least one outside system (e.g., a computer not associated with the clinical assay system) or at least one outside database (e.g., a Meditech database). This configuration allows the clinical assay system to directly report results and billing information to at least one healthcare provider, at least one third party payor (e.g., an insurance company), or to both.
Optionally, the software can be written such that the central controller performs a final check of assay results before sending to the outside system or database, halting transmission of the data and/or alerting a technician of potential problems with the results. For example, where two samples from the same patient are submitted for duplicate analyses by the clinical assay system, the interface program can include a checking scheme to ensure that the results of the duplicate analyses agree with each other. If the duplicates do not agree (e.g., where both a positive and a negative result are reported to the central controller by the clinical assay system), the interface program can halt transmission of the results so that the mass spectroscopy data can be reanalyzed or to allow the assays to be performed again.
The central controller also includes a software program for linking at least one other module of the system to the central controller (“linking software”). The module(s) also include linking software allowing the module(s) to communicate with the central controller. For example, the linking software can allow the central controller to control the module (e.g., to instruct the module as to when and whether to execute a function), and to provide to the module(s) information about the biological sample (e.g., the tests to be performed on the sample). The linking software also allows the central controller to receive information from the module(s), e.g., assay results, information about the location of the sample, and the like.
Also included within the clinical assay system is plate editor software 7. The plate editor software can be loaded on the central controller and/or on a separate computer, e.g., a second computer system. The plate editor software can receive information from an outside system or database, such as patient information, tests to be performed, billing information, etc., and create a file that maps on a hypothetical 384-well plate where each assay for each sample will be located, and links this location to all data and information associated with each assay (one sample can have several assays, e.g., 1, 2, 3, or 4 assays). In this way, the plate editor software “maps out,” in advance of processing a batch of samples through the clinical assay system, an arrangement of assays on a 384-well plate. This file can be used by other modules in the system, e.g., the sample PCR preparation system and the mass spectrometry system, to track the samples and their associated assay results as they move through the system.
Also included is a relational database 9, e.g., Oracle software. Like the plate editor software, the relational database can be loaded on the central controller and/or a separate controller, e.g., a second computer system. The relational database stores information from the plate editor and from an outside database or system (e.g., Meditech), and can be accessed by the outside system or database.
The sample transfer system 10 can include software causing it to receive at least one biological sample (e.g., a batch of samples), to obtain information about each sample (e.g., from a bar code associated with the sample), and to place an aliquot of the sample into a multiwell plate while keeping track of the location of the aliquot within the multiwell (e.g., a 96 well) plate. The software can instruct the sample transfer system to compile the information (e.g., identity of the sample and the location of the sample in the multiwell plate) into a file, which can then be transmitted to the central controller and/or other modules, e.g., the PCR preparation system.
An example of software that can be used, for example, with sample transfer system 10 is attached hereto as Appendix 1 and is described in detail below.
OutFileGenerator.exe
Laboratory software, e.g., GeminiTM (Tecan) software, allows users to control robotic and liquid handling functions and to write specific application scripts based on the user's needs. OutFileGenerator.exe is part of the Gemini program that performs specimen transfer from original bar-coded tubes into a 96-well destination plate, prior to extraction. First, all racks, test tubes, and destination plates are scanned by the barcode scanner. The positions of all racks, tubes, and destination plates, are loaded to the file C:\Program Files\Gemini\Output\” created by Gemini the function. OutFileGenerator.exe creates another file, called “outputdestID.csv” (destID=barcode of the destination plate), by rewriting the original “Output” file such that it contains the following information in a consistent order: DNA ID, DestWeIIID (well number in the destination plate), Dest ID (ID of destination plate (as determined by the barcode of the plate), SourceWell ID (position of the tube in the rack), and Source ID (barcode of the rack).
Because samples are often transferred from one plate to another during the extraction process, both plates can be assigned the same barcode number. Alternatively, a script similar to “output file generator” can be used to rename the file and destination plate ID after extraction is complete.
The PCR preparation system can include software causing it to obtain information (e.g., sample identification and assays to be performed on each sample) about each sample from the sample transfer system and the plate editor, and causing it to add specific PCR materials (e.g., specific primers, nucleotides, etc.) to each sample. In this way, the PCR preparation system is instructed as to which test(s) is to be performed on a given sample, and the PCR preparation system adds the materials appropriate for performing that test(s).
In particular and as discussed above, the software can cause the PCR preparation system to (a) dispense appropriate assay mixes; and (b) to add samples to the appropriate assay mix. Examples of programs instructing functions (a) and (b) are attached hereto as Appendix 2 and 3, respectively, and are described in further detail below.
(a) Assay Transfer.gem
Assay Transfer.gem is an exemplary Gemini program (Appendix 2) for dispensing an aliquot 3 μl, e.g., of assay mixes to PCR plates, e.g., 384-well plates, (i.e., performing function (a) as discussed above). It uses a file called “input file,” which is exported from Plate Editor MassARRAY (Sequenom). This file contains all information about the PCR plate, e.g., the location on a plate a particular assay will be run and into which a particular DNA sample should be placed, as well as the barcode of the plate. The following fields are exported from the Plate Editor and are contained in “input file”: Plate ID—PCR plate barcode number; Group ID—name of the MassARRAY file; Assay ID—name of the assay; Sample ID—specimen's barcode; and Well Position—position in the plate. Hence, each position in the plate(s) is described by the fields described above, i.e., plate ID, Assay ID, and Sample ID. This file is used in both Assay Transfergem and DNA Transfergem, which is described in further detail below.
In this Gemini program are imbedded three executable scripts written in Visual Basic (VB): PCRMix Transfer.exe, Move File Assays.exe, and MoveFileProcessed.exe. Generally, in this program, Module 1 changes the name of a letter/number well (e.g., A1, A2) description, into a well number for a 96 well plate; Module 2 performs the same function as Module 1, but for a 384 well plate; Module 3 includes comments for module 4 execution; and Module 4 generates a worklist for making the transfer of assay mixes into a 384 well plate.
Specifically, the events as dictated by the program are as follows:

    
    
        1) the barcode of the PCR plate is read by barcode reader (Gemini script);
        2) the major part of the program is run by PCRMix Transfer.exe, VB script:
        
            a) the input file is opened and the column “Well Position” containing alphanumeric characters is divided into two columns: one contains alpha-characters and the second contains numerical characters;
            b) the file is sorted by column containing numerical characters, meaning that assay mixes will be dispensed later by columns. Hence, all 8 tips will be used at the same time in most cases, speeding up the process of dispensing PCR mixes;
            c) module 2 PCR Mix takes a combination letter/number well description and converts it to a well number for a plate (e.g., A1=1);
            d) the Gemini program loads “worklist,” and Module 3 contains directions to read the .ini file which describes the location and names of: Input File=C:Gemini\Data\Assays\Input File; Worklist file=C:\Gemini\Data\Assays\assays.gwl; Source Type=Trough 1 column; Dest Plate=384 well Marsh; Volume=3 (volume of assay mix dispensed); and WashWorklist; and
            e) module 4: (1) opens the sorted Input file and parses it to determine the well into which a particular PCR mix goes into; (2) gives information about where to find the PCR Mix—the ID of the PCR Mix (assay) is written in Gemini file by naming the Trough with the PCR Mix ID; (3) directs when, where and how washes are performed. Each wash is carried out as follows: (i) aspiration of 290-400 μl of bleach; (ii) tips are immersed in the bleach for 10 seconds; (iii) bleach is dispensed into wash station; followed by (iv) three washes with water, for example: 10 ml, 5 ml, 5 ml each. Bleach washes of the tips are performed if the Mix ID is different. A full wash (both bleach and water) is always performed at the end of worklist.
        
        
        3) the data from the run is exported into an output file by the “Export Data” command in the Gemini program;
        4) the worklist is copied and saved under a barcode.txt file name (i.e., barcode of 384-well PCR plate) by Move File Assays.exe. Hence, the worklist file is emptied and ready for the next run; and
        5) the above file is moved from the original directory into an Output Processed Directory by MoveFileProcessed.exe script.
    
    


In short, the following commands are contained in the Gemini program: number of plates to be run (in Gemini); reading of barcodes (instructions in Gemini); Execute:
C:\programFiles\PCRMixTransfer\PCRMix Transfer.exe; Load Worklist;
C;Gemini\Data\Assays\assays.gwl; Execute loaded worklist; Export Data; Execute:
C:\ProgramFiles\\Project\Move File Assays.exe; and Execute:
C:\ProgramFiles\MoveFileProcessed\MoveFileProcessed.exe.
(b) DNA Transfer.gem
The DNA Transfer.gem program (Appendix 3) is an exemplary Gemini program for obtaining an aliquot, e.g., 2 μl, of DNA aspirated from DNA plate, e.g., a 96-well plate, and dispensing it to PCR plate, e.g., a 384 well plate, which contains a particular PCR mix. There are four executable scripts written in VB in this program: DNATransfer.exe; MoveFile.exe; FinalOutputConverter.exe; and CompareFiles.exe.
Generally, the program uses two files. The first, “InputFile,” is exported from the Plate Editor of MassARRAY software saved in csv format (which is the same file used in AssayTranfer.gem for dispensing the PCR mixes). This file dictates where the DNA will be dispensed in the PCR plate (e.g., 384-well plate) by giving for each specimen ID an exact destination, i.e., a specific plate and well. The second, “Output barcode#.csv,” (made on FreedomTM (Tecan) by OutFileGenerator.exe) specifies the location of DNA samples in a particular 96-well DNA plate. Based on these two files, a worklist is prepared, which dictates where the DNA samples are dispensed to the PCR plate (destination plate).
In short, the following commands are contained in this Gemini program: How many DNA plates will be run (in Gemini)?; How many PCR plates will be run (in Gemini)?; Reading the plates barcodes (instructions in Gemini); Execute:
C:\programFiles\DNATransfer\DNATransfer.exe; Loading worklist
C:\Gemini\Data\DNAtransfer\DNA list gwl; Execute loaded worklist; Export Data; Execute:
C:\programFiles\MoveExistFiles\MoveFile.exe; Execute:
C:\programFiles\FinalOutputConverter\FinalOutputConverter.exe; and Execute:
C:\programFiles\CompareFiles\CompareFiles.exe
The details of the program are provided below.

    
    
        1) At the start of the program, there are instructions written in Gemini which ask: how many DNA plates will be run? How many PCR plates will be run? Instructions for reading the barcodes of each DNA plate and PCR plate are provided.
        2) The major part of the DNA Transfer.gem is run by DNA Transfer.exe inVB script:
        
            Module 1: instructs that the ‘input file’ is opened and the column “Well Position” containing alphanumeric characters is divided into two columns: one contain alpha-characters characters and second containing numerical characters; the file is sorted by column containing numerical characters first and then by column with alpha-characters.
            Module 2: instructs conversion of combination letter/number well descriptions to a well number for a 384 well plate. (for example A1=1).
            Module 3: contains instructions to read the .ini file which describes the location and names of: DataFile-=Input File; Worklist;Source Type=96 well plate Sarstedt; Destination Plate=384-well Marsh; and Volume=2 (volume of dispensed DNA).
            Module 4: (a) contains a loop that takes each DNA ID from the ‘inputfile’ and goes through all files in the Output directory (containing Output'barcode#.csv” files) looking for the match of the ID to determine which well of the source plate from which a sample should be taken; and (b) creates a workfile containing all information needed for each DNA transfer, such as DNA ID, source well number, source plate type, source plate ID (that is a barcode of the 96-well DNA plate), volume to transfer, destination plate type, destination plate ID (barcode of 384-well PCR plate), destination well and tip number to be used; (c)
            CreatesTextFile(“C:\Gemini\Logfiles\DNAIdLog.txt”); (d) instructs when, where and how the washes are performed. Each wash is carried out as follows: (i) aspiration of 290-400 μl of bleach; (ii) tips are immersed in the bleach for 10 seconds; (iii) bleach is dispensed into wash station; followed by (iv) three washes with water, for example: 10 ml, 5 ml, 5 ml each. Bleach washes of the tips are performed if the Mix ID is different. A full wash (both bleach and water) is always performed at the end of worklist; and e) instructs termination of the run if it cannot file DNA in the outputbarcode file .csv plate, giving the message: “DNA Ids missing! See DNAIdLog, fix files, and rerun”; (f) when all DNAs are found, the worklist is made and the files Output'barcode#.csv are moved from :\Gemini\Output directory to C:\Gemini\OutputProcessed.
        
        
        3) Worklist is loaded to Gemini and executed by Gemini command.
        4) Worklist is copied and saved under ‘outbarcode.txt’ file name (barcode of 384-well PCR plate) by MoveFile.exe. Hence, the worklist file is emptied (cleaned out) and ready for the next run.
        5) FinalOutputConverter.exe module 1 converts the original output file and puts it in a readable format containing: “DNA ID,” “Destination Well ID,” “Destination plate ID,” “Source Well ID,” and “Source Plate Id.” Module 2 converts well number from integer (1) back to alphanumeric well number (A1). Module 3 scans the directory and looks for files and converts them by adding the date to the files.
        6) In addition to checking DNA IDs between two files: ‘InputFile’ and ‘BarcodeOutput file(s)’ before the transfer starts (DNATransfer.exe module 4) CompareFiles.exe compares the “InputFile” with LogFile for correctness in transfer from source 96-well plate(s) to destination 384-well plate(s). It gives an error message if DNA is found to be missing.
    
    


The primer extenstion preparation system can include similar software, i.e., software that causes it to receive information about each sample and the assays to be performed, so that the primer extension preparation system adds the specific primer extension reaction materials (e.g., specific detection extension primers, termination mixes, etc.) appropriate for performing that test(s) on the sample.
Implementation
The methods and systems described herein can use a communications network to transmit information from the system (e.g., from the central controller) to healthcare providers and/or insurance companies. These communications networks can use either wired or wireless interfaces. For example, a communications network can be the internet, or a wire or optical cable.
The new methods can be carried out using various means of data storage. For example, the information or data relating to each sample can be stored on a computer-readable medium or in a computer memory. The information can be transferred physically on diskettes or electronically, e.g., on a dedicated intranet, or on the Internet. The data can be encrypted using standard encryption software from such companies as RSA Security (Bedford, Mass.) and Baltimore®. The data can be stored in various formats, e.g., spreadsheets or databases.
The invention can be implemented in hardware or software, or a combination of both. The invention can be implemented in computer programs using standard programming techniques following the method steps and figures disclosed herein. The programs (or scripts) should be designed to execute on the various modules or in the central controller. The output information is transmitted to one or more output devices such as a printer, or a CRT or other monitor, or a web page on a computer monitor with access to a website.
Each program used in the new methods can be implemented in a procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language can be a compiled or interpreted language.
Each computer program can be stored on a storage medium or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the central controller when the storage media or device is read by the controller (or a given module) to perform the steps or procedures described herein. The system can also be considered to be implemented as a computer-readable medium, configured or encoded with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
Although any communications network can be used, the Internet provides a useful choice to transmit data. In this method, files of data are transmitted from the system to a user in encrypted form, with each party privy to the decryption technique necessary to process the particular data, ending with the completely processed data being sent to the health care provider over the Internet in a similarly encrypted manner. In this method, the entire process can be performed in minutes once the biological sample has been obtained and processed.
Electronic Data Storage and Processing
The data is typically provided in digital form that can be transmitted and read electronically, e.g., via the Internet, on diskette, or any other mode of electronic or non-electronic communication.
As used herein, “sequence information” refers to any nucleotide and/or amino acid sequence information, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, or mutated sequences. Moreover, information “related to” the sequence information includes detection of the presence or absence of a sequence (e.g., detection of a mutation or deletion), determination of the concentration of a sequence in the sample, and the like. These sequences can be read by electronic apparatus and can be stored on any suitable medium for storing, holding, or containing data or information that can be read and accessed by an electronic apparatus. Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media. The medium is adapted or configured for having recorded thereon sequence information.
As used herein, the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus; communications networks, including local area networks (LAN), wide area networks (WAN), Internet, Intranet, and Extranet; and local and distributed processing systems.
As used herein, “stored” refers to a process for encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the sequence information.
A variety of software programs and formats can be used to store the sequence information on the electronic apparatus readable medium. Any number of data processor structuring formats (e.g., text file or database) can be employed to obtain or create a medium having recorded thereon the sequence information.
By providing sequence information in computer-readable form, one can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the sequence information in readable form to compare a specific sequence with the sequence information stored within a database. Search means are used to identify fragments or regions of the sequences that match a particular sequence.
The present invention therefore provides a medium for holding instructions for performing a method for determining whether an individual has a specific disease or disorder or a pre-disposition, for a specific disease or disorder based on genetic information.
Methods of Using the Clinical Assay System
The new methods make use of the new clinical assay systems described herein. One method 26 of using an exemplary configuration of the clinical assay system is described below and illustrated in FIG. 2.
Biological samples to be processed (e.g., blood samples) typically arrive at the clinical assay system in multiple containers such as test tubes. Information about the samples (e.g., patient identification, tests to be performed, expected turn-around time) can accompany the sample and/or can be encoded within a barcode associated with the sample. Referring to FIG. 2, the information is first entered into the central controller (step 28) and/or the plate editor, e.g., using a keyboard or bar code scanner, and optionally coordinated (e.g., matched) with information about the sample received by the central controller from an outside system or database (step 30) (e.g., via a Meditech interface). Information obtained by the central controller can be transmitted to one or more modules of the clinical assay system immediately and/or at appropriate intervals during operation of the system.
The biological samples are deposited in holders in the sample transfer system. Aliquots of the biological samples are removed from the biological sample containers and deposited into multiwell plates by the sample transfer system (step 32). The sample transfer system can include a subsystem, e.g., a barcode scanner and appropriate software, capable of tracking where each aliquot of a biological sample is deposited within the multiwell plate. The information generated via the subsystem can be transmitted to the subsequent modules in the system and/or to the central controller.
The plate containing the aliquots is then transferred to the nucleic acid extraction system for DNA or total nucleic acids extraction (step 34). Following execution of the extraction process, the aliquots are transferred by the nucleic acid extraction system to a different multiwell plate.
The plate containing extracted nucleic acid samples is then transferred to the nucleic acid measurement system wherein the concentration of extracted nucleic acids in each aliquot is measured (step 36). The nucleic acid measurement system can include a subsystem, e.g., appropriate software, for assigning the data generated during the measurement process to each individual aliquot and transmitting this data to the central controller.
The plate is then transferred to the PCR preparation system. The PCR preparation system performs at least related two steps: (1) dispensing appropriate assay mixes, which include all components required for carrying out PCR reactions (e.g., buffers, dNTPs, primers, and polymerase enzyme(s)) (step 37); and (2) dispensing nucleic acid samples, e.g., control and patient DNA samples (step 38). The PCR preparation system can also include a subsystem, e.g., appropriate software, which allows the PCR preparation system to receive from the central controller information specifying which assay(s) is to be performed on a given aliquot, to locate the aliquot on the plate, and to dispense appropriate primers and nucleic acid samples based on those specifications into the aliquot.
The plate is then transferred to the theromcycling system for amplification of the target nucleic acid sequence (step 40). The PCR reactions can be performed using any appropriate thermocycling protocol, e.g., a protocol as described in the Examples section of the present application.
Following execution of the PCR reaction, the plate is transferred from the thermocycling system to the primer extension preparation system. The primer extension preparation system adds a dephosphorylating enzyme, e.g., Shrimp Alkaline Phosphatase (SAP), to each aliquot to dephosphorylate unincorporated dNTPs leftover from the PCR reaction (step 41). The plate is then optionally incubated. An extension mix, which includes the extension primer, appropriate enzymes, and a mixture of dNTPs and ddNTPs, is added to each aliquot by the primer extension preparation system (step 42). The primer extension preparation system can also include a subsystem, e.g., appropriate software, which allows the primer extension preparation system to receive from the plate editor information specifying which assay(s) is to be performed on a given aliquot, to locate the aliquot on the plate, and to dispense into each aliquot appropriate extension primers and mixtures of dNTPs and ddNTPs based to those specifications.
The plates are then transferred back to the thermocycling system, or to a second thermocycling system (e.g., a second thermocycling system contained within the primer extension preparation system), to allow the primer extension reaction to occur (step 44). After the primer extension reaction is complete, the plates are optionally transferred back to the primer extension preparation system, where resin is added by the system to the plates to remove salts, which could potentially interfere with MALDI-TOF analysis.
The plates are then transferred to the mass spectrometry preparation system. The system transfers a sample (e.g., nanoliter volume samples) of each aliquot on the plate to a chip (e.g., a silicon chip) for MALDI-TOF analysis (step 46).
Chips containing the samples are transferred from the the mass spectrometry preparation system to the mass spectrometry system. The mass spectrometry system analyzes the samples using MALDI-TOF mass spectrometry (step 48). Specifically, the mass spectrometry system analyzes extended detection extension primers on the basis of molecular weight. Software associated with the mass spectrometry system receives the raw mass spectrometry data, performs digital signal processing (e.g., de-noise, baseline determination, and data compression), analyzes the data, and calls the geneotype according to the resulting data (e.g., mass data). The software associated with the mass spectrometry system optionally receives information from the central controller and matches the data with information about the sample (e.g., patient identification and the assay performed). The data is exported from the mass spectrometry system to the relational database and/or central controller (step 49).
The finalized data, e.g., patient name, assays performed, and genotype, are exported from the relational database and/or the central controller to an outside system or database (e.g., a Meditech database) (step 50), e.g., via an interface program, e.g., automatically. The outside system or database is accessible by healthcare providers and/or third party payors. Optionally, the information exported from the central controller to the outside system or database can include billing information, e.g., billing codes for the test or tests performed, allowing direct billing of a third party payor for services rendered.
Optionally, the central controller can be programmed to include with the test results diagnosis information. Such information can be used to facilitate, or give a definitive, diagnosis. For example, with regard to infectious diseases, the diagnostic information can include an indication that a patient has been infected with a particular pathogen, the severity of the infection, the genotype of the organism (e.g., to facilitate the tailoring of a treatment, e.g., to treat a drug resistant organism with a drug to which it is sensitive), and a recommended regimen of treatment. As another example, with respect to genetic mutations that predispose an individual to genetic disease, the information can indicate whether the patient is hetero- or homozygous for the mutation, the likelihood that the patient will develop a disease or condition because of the mutation, and recommend a course of action such as a regimen of treatment or prevention. Such information can be associated with the patient data as necessary and exported from the central controller to the outside database or system.
Clinical Assays
In addition to the new system, the present invention provides novel diagnostic assays. The assays employ novel primer sequences and mass spectrometry (e.g., MALDI) to detect the presence of target nucleic acid sequences and/or sequence variations (e.g., insertions, mutations and/or polymorphisms) in biological samples. The assays can be categorized into two groups: genetic tests (e.g., to detect mutations) and infectious disease/pathogen testing (e.g., to detect the presence and/or amount of foreign, e.g., viral and/or bacterial, nucleic acids, and/or to genotype the pathogen). A general description of the assays is provided below, and several exemplary assays are described in the Examples.
Design of Primers and Specific Considerations
In one embodiment, the novel genetic assays of the present invention require primers for PCR amplification of a target sequence and detection extension primers for carrying out primer extension reactions.
A target sequence can be, e.g., a sequence that is part of a gene, or a mutation (e.g., insertions, deletions, transitions, or transversions), which is known, or suspected, to be associated with (e.g., cause, increase, or decrease the risk of) a genetic disease or disorder. A target sequence can also be a sequence that is part of a pathogen's (e.g., a virus, bacterium, or fungus) genome, which optionally is known or suspected to include variations (e.g., insertions, deletions, transitions, transversions, etc.) conferring special characteristics, e.g., possession of virulence factors and/or drug resistance, and/or which serve to differentiate among strains of the organism.
Generally, to generate amplification primers, a 100 bp stretch of nucleotides is first selected from either or both sides of a target sequence. A set of primers is then created manually and/or by using commercially available primer-generating software. An example of such a program is the Sequenom® Spectro DesignerTM program. This results in a first generation of amplification primers and appropriate detection extension primers.
The first generation of extension and/or amplification primers is analyzed using a second program, such as OLIGO® Primer Analysis Software (Macintosh, Molecular Biology Insights, Inc.), which evaluates each primer with regard to melting temperature of the primer (Tm), potential hairpins in each primer (judged by free energy (ΔG), number of nucleotides in the loop, and Tm of the hairpin), and potential for primer dimerization. This analysis results in a second generation of primers.
The second generation of amplification and/or extension primers is then compared to other known sequences, e.g., using a BLAST program (see, e.g., the National Institutes of Health's National Center for Biotechnology Information (NCBI) website on the World Wide Web at address ncbi.nlm.nih (top level domain “.gov”)). Primers that match with a sequence other than the target sequence are disregarded. Primers that match with only the target sequence are considered a third generation of amplification primers and are retained for further analysis.
All possible combinations of the third generation of amplification primers and the detection extension primers are run through the diagnostic genetic assay with positive and negative controls to identify sets of primers that are accurate in 100% of genotype calls. Amplification primers so identified are considered a fourth generation of amplification primers, and are suitable for use in the assays of the present invention.
The procedures discussed above can be modified for designing primers for a target gene when a homologous pseudogene(s) is known to be present, e.g., the glucocerebrosidase (GC) gene, which is involved in Gaucher Disease. Many commercially available software packages are not able to design specific primers when a homologous sequence to the target gene is present in the genome. Many design primers that amplify short stretches of DNA sequence (60 bp to 150 bp) because PCR is more efficient when short fragments of DNA are amplified. Where pseudogenes are present, however, the amplified fragments of DNA often must be as large as 1000 bp or more. In such cases, at least one of the amplification primer sequences directed against the functional gene must not be present in the pseudogene sequence. For example, based on a comparison of the target gene and pseudogene sequences, a primer can be designed, manually or using software, e.g., software other than SpectroDESIGNERTM, that will prime the target gene, but not the pseudogene (homologous) sequence. The extension primer can then be designed using software, e.g., SpectroDESIGNERTM. Each amplification and extend primer can then be analyzed using a program like BLAST for specificity (i.e. absence of repetitive or homologous sequences) of the sequence. At least one of the amplification primers should be specific.
In certain embodiments of the invention, duplicative confirming assays are used. In such cases, an assay is designed around a target using both the sense and antisense strand sequences, designing both forward and reverse detection extension primers. The results from both assays can be compared. Duplicative confirming assays can be beneficial, for example, if the region surrounding the target includes polymorphisms that prevent the PCR amplification and/or extension primers from working, which would cause erroneous results. They can also reduce the occurrence of mistaken calls, e.g., the calling of a heterozygote as a homozygote in an instance when one primer on one allele does not work because of a polymorphic sequence. Generally, when designing these types of confirming assays, two sets of amplification primers are prepared, each extend primer having its own pair of amplification primers. The second pair of amplification primers are designed either manually or using a program, e.g., a program other than SpectroDESIGNERTM.
In certain other embodiments, primers for targets can be designed using more than one type of software, e.g., using SpectroDESIGNER with the input of another sequence aligning software, for additional security when manipulating sequences. For example, in one assay design protocol, the sequence having the deletion/insertion is duplicated. One of the sequences is used to design a forward extension primer, while the other sequence is used to design a reverse extension primer. SpectroDESIGNERTM is used to design extension primers in the forward or reverse direction. To force SpectroDESIGNERTM to design extend primers in the forward or reverse direction, the letter “N” can be introduced into the sequence at a position 3′ to the deletion/insertion site when designing the forward extend primer, or 5′ to the deletion/insertion site when designing a reverse extension primer.
The targets for SpectroDESIGNERTM when designing primers for a deletion/insertion site is the first base in the deletion or insertion and the first base after the deletion or insertion. Often, these two nucleotide targets differ for the forward and reverse extension primers. The sequence following the target nucleotides for a forward extend primer can omit the first nucleotide after the deletion/insertion; and, similarly for the reverse extend primer, the nucleotide prior to the deletion/insertion can be omitted. For the purpose of designing the assay with a forward extension primer, in certain instances the sequence following a target should be the sequence that actually follows the deletion/insertion site in genomic DNA. In other cases, the target should be followed by the deleted/inserted sequence. Similar considerations are applicable to designing reverse primers. The choices of how to design the primers in view of these considerations can be dictated to SpectroDESIGNERTM by a second type of software, e.g., software other than SpectroDESIGNERTM. The validity of the extension primer designed by SpectroDESIGNERTM is checked by aligning the extended primer sequences (Ext1 and Ex2) with the template sequence, with and without the insertion/deletion in order to check the validity of assay design.
Novel Primers
A number of novel primers were isolated for use in the assays of the present invention. Accordingly, the present invention includes the following nucleic acid sequences, in which the MTHFR primers are used to detect mutations in the 5,10-Methylenetetrahydrofolate Reductase gene, the Fall primers are used to detect mutations in the Coagulation Factor II (FII) gene, the FaV primers are used to detect mutations in the Coagulation Factor V (FaV) gene, and the HFE and FM primers are used to detect mutations in the HFE gene. All others are used to detect the presence and amount of cytomegalovirus (CMV) in a biological sample.










MTHFR-F1
(SEQ ID NO: 1)









5′-ACGTTGGATGATGCCTTCACAAAGCGGAAG-3′



 










MTHFR-R2
(SEQ ID NO: 2)









5′-ACGTTGGATGCTTGAAGGAGAAGGTGTCTG-3′



 










MTHFR-E3
(SEQ ID NO: 3)









5′-TGCGTGATGATGAAATCG-3′



 










MTHFR-F17
(SEQ ID NO: 4)









5′-ACGTTGGATGAGTGATGCCCATGTCGGTG-3′



 










MTHFR-R18
(SEQ ID NO: 5)









5′-ACGTTGGATGCTGACCTGAAGCACTTGAAG-3′



 










MTHFR-E6
(SEQ ID NO: 6)









5′-GGAGAAGGTGTCTGCGGGAG-3′



 










MTHFR-F4
(SEQ ID NO: 7)









5′-ACGTTGGATGTCTACCTGAAGAGCAAGTCC-3′



 










MTHFR-R5
(SEQ ID NO: 8)









5′-ACGTTGGATGTCTCCCGAGAGGTAAAGAAC-3′



 










MTHFR-E11
(SEQ ID NO: 9)









5′-AACAAAGACTTCAAAGACACTT-3′



 










MTHFR-F7
(SEQ ID NO: 10)









5′-ACGTTGGATGACTACTACCTCTTCTACCTG-3′



 










MTHFR-R8
(SEQ ID NO: 11)









5′-ACGTTGGATGCTCCAGCATCACTCACTTTG-3′



 










MTHFR-E14
(SEQ ID NO: 12)









5′-GAGGAGCTGACCAGTGAAG-3′



 










FaII-F18
(SEQ ID NO: 13)









5′-ACGTTGGATGACTCATATTCTGGGCTCCTG-3′



 










FaII-R19
(SEQ ID NO: 14)









5′-ACGTTGGATGAGAGAGCTGCCCATGAATAG-3′



 










FaII-E16
(SEQ ID NO: 15)









5′-CACTGGGAGCATTGAGGCT-3′



 










FaII-F10
(SEQ ID NO: 16)









5′-ACGTTGGATGTGGAACCAATCCCGTGAAAG-3′



 










FaII-R11
(SEQ ID NO: 17)









5′-ACGTTGGATGAGAGAGCTGCCCATGAATAG-3′



 










FaII-E15
(SEQ ID NO: 18)









5′-CAATAAAAGTGACTCTCAGC-3′



 










FaV-F16:
(SEQ ID NO: 19)









5′-ACGTTGGATGAAGACCATACTACAGTGACG-3′



 










FaV-R17:
(SEQ ID NO: 20)









5′-ACGTTGGATGCATTATTTAGCCAGGAGACC-3′



 










FaV-E10:
(SEQ ID NO: 21)









5′-GACAAAATACCTGTATTCCT-3′



 










FaV-F18:
(SEQ ID NO: 22)









5′-ACGTTGGATGCTCTGGGCTAATAGGACTAC-3′



 










FaV-R19:
(SEQ ID NO: 23)









5′-ACGTTGGATGCTGAAAGGTTACTTCAAGGAC-3′



 










FaV-E9:
(SEQ ID NO: 24)



5′-GCAGATCCCTGGACAGGC-3′




 



460 F1
(SEQ ID NO: 25)









5′-ACGTTGGATGGTCGTGTATGCCACTTTGAC-3′



 










460 R2
(SEQ ID NO: 26)









5′-ACGTTGGATGTGAGGCTGTAATCGCACAGC-3′



 










460 E3
(SEQ ID NO: 27)









5′-CCACTTTGACATTACACCC-3′



 










520 F8
(SEQ ID NO: 28)









5′-ACGTTGGATGTCTTTCAGGAGACGGGTACG-3′



 










520 R9
(SEQ ID NO: 29)









5′-ACGTTGGATGAGATGAGCAGCTTCTGCAGC-3′



 










520 E10:
(SEQ ID NO: 30)









5′-TCTGCGCGAATGTTACCA-3′



 










591 F12
(SEQ ID NO: 31)









5′-ACGTTGGATGAGGCGTTGCTCTTTAAGCAC-3′



 










591 R13
(SEQ ID NO: 32)









5′-ACGTTGGATGAGTGCGTGAGCTTACCGTTC-3′



 










591 E14
(SEQ ID NO: 33)









5′-TTAAGCACGCCGGCGCGG-3′



 










607 F28
(SEQ ID NO: 34)









5′-ACGTTGGATGTCATTTGCGCCGCCAGAATG-3′



 










607 R29
(SEQ ID NO: 35)









5′-ACGTTGGATGTTGCTCTTTAAGCACGCCGG-3′



 










607 E30
(SEQ ID NO: 36)









5′-GCCGCCAGAATGAGCAGA-3′



 










CV-F31
(SEQ ID NO: 37)









5′-ACGTTGGATGGAGACGACGTGGACGGCAC-3′



 










CV-R32
(SEQ ID NO: 38)









5′-ACGTTGGATGCGTGCTTGGACACGCGACTT-3′



 










CV33 E
(SEQ ID NO: 39)









5′-CTCCTTGTCCGAAGCCGC-3′



 










HFE-F1
(SEQ ID NO: 40)









5′-ACGTTGGATGATGACCAGCTGTTCGTGTTC-3′



 










HFE-R2
(SEQ ID NO: 41)









5′-ACGTTGGATGTCTACTGGAAACCCATGGAG-3′



 










FM3-E
(SEQ ID NO: 42)









5′ CTCCACACGGCGACTCTCAT-3′



 










HFE-F7
(SEQ ID NO: 43)









5′-ACGTTGGATGTTCATGGGTGCCTCAGAGC-3′



 










HFE-R8
(SEQ ID NO: 44)









5′-ACGTTGGATGCCACATCTGGCTTGAAATTC-3′



 










HFE-E3
(SEQ ID NO: 45)









5′ CAGCTGTTCGTGTTCTATGAT 3′



 










HFE-F4
(SEQ ID NO: 46)









5′-ACGTTGGATGTGGATAACCTTGGCTGTACC-3′



 










HFE-R5
(SEQ ID NO: 47)









5′-ACGTTGGATGTATCACAATGAGGGGCTGATC-3′



 










FM6-E
(SEQ ID NO: 48)









5′ GCCTGGGTGCTCCACCTGG 3′



 










HFE-F9
(SEQ ID NO: 49)









5′-ACGTTGGATGTAATGGGGATGGGACCTACC-3′



 










HFE-R10
(SEQ ID NO: 50)









5′-ACGTTGGATGTGCTCTCATCAGTCACATACC-3′



 










HFE-E6
(SEQ ID NO: 51)









5′ GGGAAGAGCAGAGATATACGT 3′



 










HFE-F1
(SEQ ID NO: 52)









5′-ACGTTGGATGATGACCAGCTGTTCGTGTTC-3′



 










HFE-R2
(SEQ ID NO: 53)









5′-ACGTTGGATGTCTACTGGAAACCCATGGAG-3′



 










HFE S65C_E1
(SEQ ID NO: 54)









5′ GGGCTCCACACGGCGAC 3′



 










HFE-F7
(SEQ ID NO: 55)









5′-ACGTTGGATGTTCATGGGTGCCTCAGAGC-3′



 










HFE-R8
(SEQ ID NO: 56)









5′-ACGTTGGATGCCACATCTGGCTTGAAATTC-3′



 










HFE S65C_E5
(SEQ ID NO: 57)









5′ TTCGTGTTCTATGATCATGAG 3′



 










CMV560-S:
(SEQ ID NO: 58)









5′-ACGTTGGATGCGCTTCTACCACGAATGCTC-3′



 










CMV560-L:
(SEQ ID NO: 59)









5′-ACGTTGGATGATAAATACAGCCCGTCGCTC-3′



 










CMV560-E
(SEQ ID NO: 60)









5′-CTTTCTGACGTATTCGTGCAGCAT-3′






Additional primers are set forth in FIGS. 15A-15GG, which includes a listing of the target gene, amplification primers and extension primers for that target, and the analytes.
Assays
In general, an assay of the present invention can be performed as follows. A biological sample is obtained. As used herein, a “biological sample” is material obtained from any living source (e.g. human, animal, plant, bacteria, fungi, protist, virus) that can be solid material (e.g. tissue, cell pellets, biopsies, fecal matter, nucleic acid samples) and/or fluids (e.g., urine, blood, saliva, sputum, amniotic fluid, mouth wash).
If necessary (i.e., when the starting material is an intact biological sample and not previously-extracted nucleic acids), nucleic acids are extracted from the biological sample. Extractions can be performed using any art-known method, the choice of which will depend, e.g., on the biological sample from which the nucleic acids are to be isolated. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (see, e.g., Rolff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)). There are numerous kits available for extracting nucleic acids from samples, e.g., the MagneSil® ONE, the Fixed Yield Blood Genomic System (Promega), and kits manufactured by Qiagen.
The target gene is then amplified via polymerase chain reaction (PCR) using fourth generation primers designed as described herein. Procedures for performing PCR are well known in the art, and are described in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989. Where the nucleic acids to be analyzed are RNA (e.g., viral RNA), the assays of the present invention can be adapted to include reverse transcriptase PCR (RT-PCR) reactions, if necessary.
In certain embodiments of the invention, an oligo base extension method is then used to detect mutations in, or the presence of, a target nucleic acid. The method is based on the extension of a detection primer (i.e., a detection extension primer designed as described above) that anneals adjacent to a variable nucleotide position on the amplified target DNA, using a DNA polymerase, a mixture of dNTPs (e.g., 3 dNTPs), and the missing dideoxy nucleotides (e.g., one missing dideoxy nucleotide). Any art known primer extension method can be utilized.
The sample is then analyzed using mass spectrometry. Any mass spectrometer format can be used to analyze the samples, e.g., matrix assisted laser desorption ionization (MALDI), electrospray (ES), ion cyclotron resonance (ICR) and Fourier Transform. Various mass analyzers can be used, e.g., magnetic sector/magnetic deflection instruments in single or triple quadrupole mode (MS/MS), Fourier transform, and time-of-flight (TOF) configurations as is known in the art of mass spectrometry. In an embodiment of the present invention, samples are analyzed using MALDI-TOF mass spectrometry.
The molecular weight of extended primers is determined by mass spectrometry, and a change in the molecular weight of the extended primer, as compared to a control, indicates the presence of the target sequence and/or whether a mutation (e.g., insertion or deletion), is present in the target sequence. The results are analyzed using methods described in the Examples section below, and an analysis report is generated.
In other embodiments of the present invention, the assay described above is modified to include, e.g., homogenous MassCLEAVAGETM (hMC) reactions (Sequenom GmbH, Hamburg, Germany). During PCR amplification of the target region, a T7 promoter for in vitro transcription is introduced to the 5′ end of one strand of the amplicon. One PCR is performed with a T7 promotor in the forward primer to mediate transcription of the forward DNA strand, and a second PCR is performed with the T7 promotor incorporated in the reverse primer to mediate transcription of the reverse DNA strand. Following the PCR reaction, SAP is added to each reaction to degrade any unincorporated nucleotides from the PCR reactions. For in vitro transcription production, each PCR reaction is split into two transcription reactions and performed. Next, base-specific cleavage using RNase A is conducted, yielding fragmented RNA molecules.
The final steps in the hMC reaction are sample conditioning and MALDI-TOF MS followed by spectra pattern analysis. The hMC reaction is a high throughput tool for sequence variation identification by comparative sequence analysis. The principle behind it is to generate signal patterns that are specific to a reference sequence in which changes, when compared to a sample sequence, can be tracked as a sequence variation in the form of SNPs or insertion/deletions.
Uses of Assays and the Clinical Assay System
The assays described above can be used to diagnose genetic diseases (or a patient's or subjects increased risk therefore), to identify foreign sequences (e.g., viral sequences) incorporated into a target gene, and for infectious disease/pathogen testing. The term “subject” as used herein, refers to an animal, or a human, and includes, but is not limited to, mammals, e.g., primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep, and goats. Agricultural analyses, e.g., analysis of biological samples from plants can also be made with the new systems and methods.
For example, the assays can be adapted to diagnose any of the more than 3000 genetic diseases or disorders currently known (e.g. hemophilias, Gaucher's disease, thalassemias, Duchenne Muscular Dystrophy (DMD), Huntington's Disease (HD), Alzheimer's Disease and Cystic Fibrosis (CF)). and lysosomal and metabolic diseases.
The assays can also be used to diagnose chromosomal abnormalities such as Trisomy 21 (Down's syndrome), Trisomy 13 (Platau Syndrome), Trisomy 18 (Edward's Syndrome), Monosomy X (Turner's Syndrome) and other sex chromosome aneuploidies such as Klienfelter's Syndrome (XXY), which result in birth defects, and to determine whether an individual has a genetic mutation(s) that predispose the individual to any of a number of disorders such as diabetes, arteriosclerosis, obesity, various autoimmune diseases, stroke, and cancer (e.g. colorectal, breast, ovarian, lung); chromosomal abnormality (either prenatally or postnatally); or a predisposition to a disease or condition (e.g. obesity, atherosclerosis, cancer, venous thromboembolism (VTE), neural tube defects (NTD), and the like) and lysosomal and metabolic
The assays can also be used in the detection of “DNA fingerprints,” e.g., polymorphisms, such as “microsatellite sequences,” which are useful for determining identity or heredity (e.g. paternity or maternity).
Further, the assays can be used to detect or quantify in a biological sample nucleic acid sequences that are specific to infectious organisms. Such assays are useful for diagnosing or monitoring infection. Examples of disease-causing viruses that infect humans and animals and that may be detected by the disclosed processes include: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, see Ratner, L. et al., Nature, 313: 227-284 (1985); and Wain Hobson, S. et al., Cell, 40: 9-17 (1985)); HIV-2 (see Guyader et al., Nature, 328: 662-669 (1987); European Patent Publication No. 0 269 520; Chakraborti et al., Nature 328: 543-547 (1987); and European Patent Application No. 0 655 501); and other isolates, such as HIV-LP (International Publication No. WO 94/00562 entitled “A Novel Human Immunodeficiency Virus”; Picornaviridae (e.g., polio viruses, hepatitis A virus, (Gust, I. D., et al., Intervirology, Vol. 20, pp. 1-7 (1983); entero viruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses), and Epstein-Barr virus (EBV).
Examples of infectious bacteria include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelli. 
Examples of infectious fungi include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. Other infectious organisms (i.e., protists) include: Plasmodium falciparum and Toxoplasma gondii. 
FIGS. 15A-15GG describes a number of specific genetic targets that can be assayed according to the invention, as well as exemplary primers that can be used in those assays. For example, exemplary primers are provided for the following genetic targets: 5,10-Methylenetetrahydrofolate Reductase (MTFR); Coagulation Factor II; Coagulation Factor V; hemochromatosis (HFE); and a glucocerebrosidase (GC). fibroblast growth factor receptor 3; aspartoacylase; Glucocerebrosidase; Coagulation Factor VII; Fanconi Anemia, Complementation Group C (FANCC); inhibitor of kappa light polypeptide gene enhancer in b cells, kinase complex-associated protein; acid sphingomyelinase; hexosaminidase; angiotensin i-converting enzyme; adenylate cyclase 9; apolipoprotein A-1; apolipoprotein E; endothelial leukocyte adhesion molecule 1; fc fragment of IGG, low affinity Ia, receptor; fibrinogen beta chain; coagulation factor II, factor XIII; guanine nucleotide-binding protein beta-3; integrin, alpha-2, glycoprotein Ia/Iia; glycoprotein Ib, platelet, alpha polypeptide; intercellular adhesion molecule 1; glycoprotein Ia/IIa (a2), integrin, alpha-2; platelet glycoprotein Iib, integrin, alpha-2b; glycoprotein IIb/IIIa, integrin, beta-3;3-hydroxy-3-methylglutaryl-coa reductase; lymphocyte adhesion molecule 1; methylene tetrahydrofolate reductase; plasminogen activator inhibitor 1; platelet alpha-granule membrane protein; transforming growth factor-beta receptor, type III; thrombomodulin; tumor necrosis factor; vascular cell adhesion molecule; coagulation factor II receptor; glycoprotein VI, platelet; purinergic receptor P2Y, g protein-coupled, 1; purinergic receptor P2Y, G protein-coupled, 12; prostaglandin-endoperoxide synthase 1; prostaglandin-endoperoxide synthase 2; thromboxane A2 receptor, platelet; and thrombospondin I. The figure lists the disorder associated with the mutation; the gene information (e.g., gene number, symbol, and name), the target mutation, the termination mix that can be used in and assay, the amplification primers, the extension primers, the analytes, and the extended primers (Ext 1, 2, and 3, i.e., the extension primer as it would appear after extension).
The invention will be further described in the following examples, which do not limit the scope of the invention defined by the claims.
EXAMPLES
Example 1
Screen for 5,10-Methylenetetrahydrofolate Reductase (MTHFR) Mutations
Introduction
In one exemplary assay, a biological sample (e.g., a blood sample) is tested for the presence or absence of mutations in the MTHFR gene. MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine. MTHFR plays a key role in folate metabolism by channeling one-carbon units between nucleotide synthesis and methylation reactions. Severe enzyme deficiency leads to hyperhomocysteinemia and homocystinuria, with altered folate distribution and a phenotype that is characterized by damage to the nervous and vascular systems.
Two common mutations that can occur in MTHFR include MTHFR C677T (or Ala222Val) and MTHFR A1298C (or Glu429Ala). Assays to detect these mutations are described below. The C677T mutation is common in the general population, with a variable frequency depending on the subpopulation studied. In Caucasians of European descent, the frequency of homozygotes for this mutation ranges from approximately 5% (in Dutch and Finnish populations) to 12-15% (in French Canadian and other European populations). African Americans have a low frequency of 1.4% The A1298C mutation is also common in the general population with an allele frequency estimated at 0.33.
Individuals homozygous for the C677T mutation have significantly elevated plasma homocysteine levels. It has been suggested that the 677T variant of the MTHFR gene is a genetic risk factor for preeclampsia. From studies of the C677T mutation in cardiovascular patients and controls, investigators have concluded that homozygosity for this frequent mutation in the MTHFR gene is associated with a 3-fold increase in risk for premature cardiovascular disease. The association was stronger in homozygotes than in heterozygotes. Therefore, the C677T polymorphism may be a risk factor for coronary artery disease.
Currently, at least 22 other mutations in MTHFR gene are known and assays similar to those described herein can be designed to detect those mutations as well.
Gene Information
Gene Locus: 1p36.3.
Gene Symbol: MTHFR.
Gene Product: 5,10-METHYLENETETRAHYDROFOLATE REDUCTASE.
Mutations: MTHFR C677T, Ala222Val, FR A1298C, Glu429Ala.
Gene OMIM number: 607093
mRNA NCBI Acc Nos.: XM—030156 5,10-methylenetetrahydrofolate reductase mRNA; AJ237672 Homo sapiens mRNA for 5,10-methylenetetrahydrofolate reductase mRNA; NM—005957 Homo sapiens 5,10-methylenetetrahydrofolate reductase (NADPH) (MTHFR) mRNA; U09806 Synthetic construct methylenetetrahydrofolate reductase (MTIFR) mRNA, complete cds. (Goyette, 1994 Frosst 1995)
Genomic Acc No.: GA_x5L2HTU1V1E CELERA DataBase
Clinical Significance
The test can be used to screen for the MTHFR gene mutation before surgery, which may identify patients at an increased risk of graft thrombosis. The MTHFR mutation assay is also useful for patients with early-onset arteriosclerotic vascular disease or thrombosis, particularly those with hyperhomocysteinemia or significant family histories. The clinical scenario should be considered carefully, since additional genetic and non-genetic factors are important in the development of cardiovascular disease. MTHFR mutation analysis is also useful for patients with variable or ambiguous homocysteine levels, since plasma levels are dependent on nutritional status or sample handling before testing. MTHFR mutational analysis can also be offered for patients with a neural tube defect (NTD). The MTHFR mutation assay is also useful in the elderly and in patients with idiopathic venous thromboembolism (VTE) disease and arterial disease.
MTHFR-C677T E3 Assay
This section describes the amplification and detection extension primers for the MTHFR-C677T E3 Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Table 1, below.

























Amplification Primers*:










MTHFR-F1
(SEQ ID NO: 1)









5′-acgttggatgATGCCTTCACAAAGCGGAAG-3′



 










MTHFR-R2
(SEQ ID NO: 2)









5′-acgttggatgCTTGAAGGAGAAGGTGTCTG-3′






Resulting Amplicon length: 89 bp

    
    
        * amplification primers have the 10-mer tag specific for the homogeneous MassEXTENDTM assay (hME) method: acgttggatg.
    
    


Detection Extension Primer:










MTHFR-E3
(SEQ ID NO: 3)









5′-TGCGTGATGATGAAATCG-3′






Termination mix: ddA/ddC/ddT







TABLE 1







Sequence, length and mass of


extension primer, extended primer


(G and A) and pause product for MTHFR-C677T










Allele
Sequence
Length
Mass





MTHFR-E3
TGCGTGATGATGAAATCG
18 bp
5578.6


 


T mutant allele
TGCGTGATGATGAAATCGA
19 bp
5875.9


(ext A)





 


C wild type
TGCGTGATGATGAAATCGGC
20 bp
6181  


allele (ext G)





 


Contaminant


5907.9


(pausing peak)





SEQ ID NOS.: 806, 807, and 808)






MTHFR C677T E6 Assay

This section describes the amplification and detection extension primers for the MTHFR C677T E6 Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Table 2, below.

























Amplification Primers*










MTHFR-F17
(SEQ ID NO: 4)









5′-acgttggatgAGTGATGCCCATGTCGGTG-3′



 










MTHFR-R18
(SEQ ID NO: 5)









5′-acgttggatgCTGACCTGAAGCACTTGAAG-3′






Amplicon length: 122 bp

    
    
        * amplification primers have the 10-mer tag specific for the hME method:acgttggatg.
    
    


Detection Extension Primer:










MTHFR-E6
(SEQ ID NO: 6)









5′-GGAGAAGGTGTCTGCGGGAG-3′






Termination mix: ddA/ddC/ddG







TABLE 2







Sequence, length and mass


of extension primer, extended primer


(G and A) and pause product for MTHFR-C677T










Allele
Sequence
Length
Mass





MTHFR-E6
GGAGAAGGTGTCTGCGGGAG
20 bp
6303.1


 


T mutant
GGAGAAGGTGTCTGCGGGAGTC
22 bp
6880.5


allele (ext A)





 


C wild type
GGAGAAGGTGTCTGCGGGAGC
21 bp
6576.3


allele (ext G)





 


Contaminant


5907.9


(pausing peak)





[SEQ ID NOS: 811, 812, and 813)






MTHFR-A1298C Assay

This section describes the amplification and detection extension primers for the MTHFR-A1298C assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Table 3, below.

























Amplification Primers*











MTHFR-F4
(SEQ ID NO: 7)









5′-acgttggatgTCTACCTGAAGAGCAAGTCC-3′



 










MTHFR-R5
(SEQ ID NO: 8)









5′-acgttggatgTCTCCCGAGAGGTAAAGAAC-3′






Amplicon length: 109 bp

    
    
        * amplification primers have the 10-mer tag specific for the hME method:acgttggatg (SEQ ID No.816)
    
    


Detection Extension Primer:












MTHFR-E11
(SEQ ID NO: 9)











5′-AACAAAGACTTCAAAGACACTT-3′






Termination mix: ddA/ddC/ddT







TABLE 3







Sequence, length and


mass of extension primer, extended


primers and pause product for MTHFR-A1298C E11:










Allele
Sequence
Length
Mass





MTHFR-E11
AACAAAGACTTCAAAGACACTT
22 bp
6704.4 


 


A mutant
AACAAAGACTTCAAAGACACTTT
23 bp
6992.60


allele





 


C wild type
AACAAAGACTTCAAAGACACTTGC
24 bp
7306.80


allele





 


Contaminant


7033.60


(pausing





peak)





(SEQ ID NOS: 817, 818, and 819)






MTHFR A1298C E14 Assay

This section describes the amplification and detection extension primers for the MTHFR A1298C E14 Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Table 4, below.

























Amplification Primers*











MTHFR-F7
(SEQ ID NO: 10)









5′-acgttggatgACTACTACCTCTTCTACCTG-3′



 










MTHFR-R8
(SEQ ID NO: 11)









5′-acgttggatgCTCCAGCATCACTCACTTTG-3′






Amplicon length: 159 bp

    
    
        * amplification primers have the 10-mer tag specific for the hME method:acgttggatg.
    
    


Detection Extension Primer












MTHFR-E14
(SEQ ID NO: 12)











5′-GAGGAGCTGACCAGTGAAG-3′






Termination mix: ddC/ddG/ddT







TABLE 4







Sequence, length and mass of


extension primer, extended primers


and pause product for MTHFR-A1298C E14










Allele
Sequence
Length
Mass





MTHFR-E14
GAGGAGCTGACCAGTGAAG
19 bp
5926.90


 


A mutant
GAGGAGCTGACCAGTGAAGAAAG
23 bp
7179.70


allele





 


C wild type
GAGGAGCTGACCAGTGAAGC
20 bp
6200.10


allele





 


Contaminant


6240.10


(pausing





peak)





(SEQ ID NOS: 822, 823, and 824)






Testing Procedure

This section generally describes procedures useful in the MTHFR assays.
Specimens: Whole blood samples were collected in the same manner routinely used for any laboratory test. Exemplary test results, with specific numbers of samples, are provided below in Tables 12, 13, 14, and 15.
Specimen Storage and Stability

    
    
        1. After arrival, blood was extracted on the same or next day. Specimen was not allowed to remain at +15° C. to +30° C. longer than 8 hours. If assays were not completed within 8 hours, specimen was stored at +2° C. to +8° C.
        2. Tubes of blood should were kept closed at all times in a vertical, stopper-up position.
        3. Blood specimen were not frozen.
    
    


Sample Volume
The minimum volume was 1.0 mL of sample.
Criteria for Unacceptable Specimens

    
    
        Unlabeled specimens.
        Insufficient quantity.
        Clotted specimen.
    
    


Specimen Handling
Blood samples were received with the lab barcode identification numbers. Sample identification is ensured thought out the process by the tracking system on Freedom, BioRobot MDx, Genesis, and MassARRAY systems.
Procedures Involved in Assays:
Nucleic acids extraction, nucleic acid concentration measurement, Uniplex and Multiplex PCR Procedure on GenesisTM in conjunction with FreedomTM and MassARRAYTM software, Homogeneous MassEXTENDTM Reaction using the MassARRAYTM System.
Quality Control
PCR primers were analyzed ensure specificity for the prothrombin gene in the BLAST search and that the PCR product did not show allelic dropout. Validation of each assay was accomplished.
Four types of control material were analyzed with each PCR plate: (A) blank control (water), (B) DNA negative control (wild type), (C) heterozygous positive controls and (D) positive homozygous mutant. In each run at least two samples of each control were used if available. More frequent use of controls or the use of additional controls was left to the discretion of the user based on work load and work flow. Tables 5 and 6, below, provide a list of control DNAs used in the presently described assays.







TABLE 5





Controls used in MTHFR- C677T assay




















39354
T
MTHFR
homozygous



GM881
T
MTHFR
homozygous



GM1278
C
MTHFR
negative- wild



NA1464
C
MTHFR
negative- wild



GM779
C
MTHFR
negative- wild



NA1465
C
MTHFR
negative- wild



GM972
CT
MTHFR
heterozygo



NA0375
CT
MTHFR
heterozygo



NA1359
CT
MTHFR
heterozygo



NA1464
CT
MTHFR
heterozygo



NA1470
CT
MTHFR
heterozygo



NA1600
CT
MTHFR
heterozygo



NA1602
CT
MTHFR
heterozygo

















TABLE 6





Controls used in MTHFR-A1298C assay




















GM7798
C
MTHFRA1298C
homozygous mutant



GM8810
A
MTHFRA1298C
negative- wild type



NA16028
A
MTHFRA1298C
negative- wild type



GM12783
AC
MTHFRA1298C
heterozygous



NA14641
AC
MTHFRA1298C
heterozygous



NA16000
AC
MTHFRA1298C
heterozygous










PCR Procedure
The master PCR mix was made, biannually or as necessary, in 11.954 ml total volume and aliquoted into 47 Sarstedt tubes, 250 II each. The composition of the PCR master mix is provided in Table 7, below.







TABLE 7







Composition of master PCR mix













Volumes used to



Stock
Final
make master PCR


Reagent
Concentration
Concentration
mix














Water
N/A
N/A
8,514
μl


Buffer/MgCl2
15 mM (10×)
1.5 mM
2,150
μl


MgCl2
25 mM (10×)
2.5 mM
860
μl


dNTPs
10 mM (50×)
200 μM
430
μl


Total volume


11,954
μl









The 5 μM primer mix was made by diluting 10 times 50 μM stock primers. The volumes used to make 2000 μl of 5 μM primer mix is shown in Table 8, below. The primer mix was aliquoted to 10 Sarstedt tubes, 200 μl each.







TABLE 8







Composition of 5 μM Primer Mix













Volumes



Stock
Concentration
used to make


Reagent
Concentration
in the mix
5 μM mix














Water
N/A
N/A
1,200
μl


Forward
50 μM
5 μM
200
μl


primer


Reverse
50 μM
5 μM
200
μl


primer


Total Volume


2000
μl









The composition of the PCR reaction is shown in Table 9. To calculate the volumes per run, the volumes per reaction of PCR mix, Primer Mix, and HotStarTaqTM need to be multiplied by number of reactions and then by 1.5. Table 9 shows an example of the calculations per 384 reactions. Increasing the final volume by 50% is to account for pipetting losses and ‘dead’ volume when using the robotics.







TABLE 9







Composition of single PCR reaction


and PCR reaction for 384-well plate












Stock


Volume per



Concen-
Final
Reaction
384 reactions


Reagent
tration
Concentration
Volume
and ×1.5















PCR mix per
10×
1×
2.78 μl
1601.28
μl


reaction


Primer Mix
5 μM
200 nM
 0.2 μl
115.2
μl


(F + R)
(40×)


HotStarTaq
5 units/μl
0.1 unit per
0.02 μl
11.52
μl



(50×)
reaction




TOTAL volume


3.00 μl
1728
μl









3 μl of PCR mix were dispensed into each well of the 384-well plate. The protocol “PCR Procedure” (MD008) was utilized. 2 μl of DNA (2.5 ng/μl) were added to each well. The standard PCR program on the PCR machine was run:


















95° C.
15 minutes





95° C.
20 seconds



56° C.
30 seconds
 {close oversize brace} 
45 cycles



72° C.
 1 minute



72° C.
 3 minutes



 4° C.
forever (hold)










Dephosphorylation Procedure
SAP (Shrimp Alkaline Phosphatase) was diluted according to Table 10, multiplying the volumes by the number of the samples and then by 1.5.







TABLE 10







Composition of SAP mix















Volume per



Stock
Final
Reaction
384-well


Reagent
Concentration
Concentration
Volume
plate















Nanopure
N/A
N/A
1.53 μl
881.3
μl


Water


hME buffer
10×
1× *
0.17 μl
97.9
μl


SAP
1 unit/μl
0.15 units/μl
0.30 μl
172.8
μl


TOTALS:


2.00 μl
1152.00
μl









2 μl of the diluted Shrimp Alkaline Phosphatase were dispensed to each well (Refer to MassARRAY procedure MD 009). The SAP PCR program was run on the PCR machine:
















37° C.
20 minutes



85° C.
 5 minutes.



 4° C.
forever (hold)










Homogeneous Massextend (hME) Reaction Procedure
The hME cocktail was prepared according to Table 11. Multiply one reaction volume by the number of samples and then by 1.38.







TABLE 11







Components of hME cocktail












Stock
Final

Volume per



Concen-
Concen-
Reaction
384-well


Reagent
tration
tration
Volume
plate*
















Nanopure
N/A
N/A
1.674
μl
887.09
μl


Water


hME Mix (10×
10×
1×
0.2
μl
105.98
μl


buffer


w/2.25 mM


d/ddNTPs)


Extension
50 μM
2.7 μM
0.108
μl
57.23
μl


primer


Thermo
32 units/μl
0.063 unit/μl
0.018
μl
9.54
μl


Sequenase&





TOTALS:


2.0
μl
1059.84
μl





*Volumes are for a 384- well microplate and include a 38% overage to account for possible pipetting loss and dead volume in the 96-well microtiter plate used on the Multimek.


&ThermoSequenase is stored at (−20)° C. until it is added to the reaction cocktail.






2 μl of the hME cocktail mix were dispensed to the appropriate wells containing 7 μl dephosphorylated PCR product. Refer to MassARRAY procedure MD008. The Extension Reaction program was run on the PCR machine:


















94° C.
2 minutes





94° C.
5 seconds



52° C.
5 seconds
 {close oversize brace} 
45 cycles



72° C.
5 seconds



 4° C.
forever (hold)










Spectroclean Resin Clean Up Procedure
Using the SpectroClean plate, transfer resin into the 96-well Marsh plate. Run the hME cation Cleanup method on the Multimeck. Refer for details to MassARRAY protocol MD008.
Results Analysis
The Genotyper® Analyzer software reads the spectrum generated for each sample by the mass spectrometer, and uses a 3-parameter model to calculate the significance of each putative genotype:

    
    
        1. Conservative: The most conservative set makes no error in reading the data.
        2. Aggressive: The most aggressive set makes the most errors (still less than 1 percent).
        3. Moderate: The moderate set is a compromise between the two extremes.
    
    


The interpretation of the results is as follows: If the results of two assays for the same mutation are called (i.e., interpreted) identically by conservative or moderate calls or conservative and aggressive calls, the result is acceptable. If the assays are called identically by moderate and aggressive calls, the test needs to be looked at by the director or the designee. If the assays are called only by aggressive calls, the test needs to be repeated. If there is a call on one but not the other assay, the “non call” assay needs to be repeated.
The non-calls can be further categorized into the following groups: (a) Low Probability: Implies that the spectrum in question contains peaks that fail any criteria of significance even for the aggressive parameter set; (b) Conflict: Implies that there is more that one read or spectrum corresponding to one sample, and these reads give rise to different and conflicting genotypes; (c) Bad spectrum: The spectrum for the sample doesn't exist above noise level; and (d) Bad Assay: results of analyst or operator input errors in defining the assays. The most common errors are mass values of analytes or contaminants that are out of range of the spectrum, or contaminants and analytes having the same mass; and (e) user call: The analyst or operator select a genotype in the table and performs a manual call.
In the presently described examples, each sample will have at least two different assays that together will confirm, e.g., the presence of a mutation. However, in many instances, accurate results can be obtained using one assay per sample and, therefore, performing two different assays is optional.
FIG. 3 is a mass spectrum of a heterozygous “TC” allele (heterozygous positive) generated using a screen for a C677T mutation in the 5,10-Methylenetetrahydrofolate Reductase (MTFR) gene. Exemplary test results are provided in Tables 12, 13, 14, and 15, below.







TABLE 12







MTHFR C677T E3















Low



Conserv-


Proba-


Source of DNA
ative
Moderate
Aggressive
bility





Associated Regional and
147
3
0



University Pathologists


(ARUP) DNAs +


Coriel Institute DNAs
















TABLE 13







MTHFR C677T E6















Low


Source of DNA
Conservative
Moderate
Aggressive
Probability





ARUP DNAs +
150
2
0



Coriel DNAs
















TABLE 14







MTHFR A1298C E11















Low


Source of DNA
Conservative
Moderate
Aggressive
Probability





ARUP DNAs +
125
26
1



Coriel DNAs
















TABLE 15







MTHFR A1298C E14















Low


Source of DNA
Conservative
Moderate
Aggressive
Probability





ARUP DNAs +
89
53
6



Coriel DNAs









The results of the assays were 100% in agreement with results from Associated Regional and University Pathologists (ARUP). In both (E3 and E6) MTHFR-C677T assays, negative controls (homozygous wild type) GM12783, GM7798, NA14641 (Coriell) were homozygous CC, positive heterozygous controls NA13591, NA14646, NA14702, and NA16000 (Coriell) were TC, and, positive homozygous control NA8810 (Coriell) was TT. In both MTHFR-A1298C assays, negative control (homozygous wild type) GM7798 (Coriell) was homozygous CC, positive heterozygous controls NA14641 and NA 16000 (Coriell) were both AC, and positive homozygous control NA 16028 (Coriell) was AA. As indicated in Tables 12, 13, 14, and 15, above, the majority of geneotype calls (i.e., the genotype called by the software for each sample) for each assay were conservative calls, indicating a high level accuracy.
Example 2
Screen for Coagulation Factor II (FII) G20210A Mutations
Mutation G20210A in the prothrombin gene is associated with hyperprothrombinemia and increased risk of venous thromboembolism (VTE). It has been established that the FII G20210A mutation increases the risk of VTE 2-5 fold. The 20210A prothrombin allele also represents an inherited risk factor for acute coronary syndrome among patients who have limited extent of coronary disease at angiography or who lack major metabolic and acquired risk factors.
Mutation G20210A involves the last nucleotide of the transcribed prothrombin RNA. The mechanism by which prothrombin levels are altered is unknown. One explanation is that increased adenylation and increased mRNA stability are involved.
In southern Europe, the prevalence of the G20210A mutation is 3.0 percent (CI95 2.3 to 3.7%), nearly twice as high as the prevalence in northern Europe 1.7% (CI95 1.3 to 2.2%), the highest of 7.8% reported in a Greek-Cypriot population. In the United States, the overall prevalence estimates are between 1% and 2%, although this is highly dependent.on race. The mutation is fairly uncommon in African Americans ( ̃0.2%), and is also rarely seen in Asians and Native Americans.
At least 28 missense mutations, one insertion, one deletion, and one substitution (in the regulatory region) are known to occur in the prothrombin gene. Accordingly, assays similar to those described herein can be designed to detect those mutations as well.
Gene Information
Gene Locus: 11p11-q12
Gene Symbol: F2, PRT
Gene Product: COAGULATION FACTOR II, prothrombin
Mutations: G20210A
Mutation Frequency: The 20210G-A variation in the prothrombin gene is predominant only in the Caucasian population, among whom the prevalence of heterozygous carriers varies between 1% to 8%, and depends on the geographical location and ethnic background.
OMIM number: 176930
mRNA NCBI Acc No.: NM—000506 coagulation factor II mRNA; M17262. Human prothrombin mRNA
Genomic Acc No.: AF478696 H.coagulation factor II (thrombin) (F2) gene.
Clinical Significance
Prothrombin (G20210A) testing can be recommended, for example, in the following cases: when inherited thrombophilia is suspected; when the testing for factor V Leiden muatation is recommended; in patients with a history of recurrent VTE; in patients with a first episode of VTE before the age of 50 years; in patients with a history of an unprovoked VTE at any age; where thrombosis occurs in unusual anatomic sites, such as cerebral, mesenteric, portal, or hepatic veins; where there is a first-degree relative with VTE; in patients with a first VTE related to pregnancy, the puerperium, or oral contraceptive use; where there is a history of VTE related to pregnancy loss during the second or third trimesters; in young women smokers (age <50 years) with an MI; in older patients (age >50 years) with a first provoked VTE event in the absence of a cancer or an intravascular device; where there is a first VTE related to serum estrogen receptor modifier or tamoifen; in selected cases of women with unexplained severe preeclampsia, placental abruption, or intrauterine growth restriction; in asympthomatic adult family members of probands with known prothrombin G20210A mutations, especially those with a strong family history of thrombosis at young age; and asymptomatic female family members who are pregnant or are considering oral contraceptives or pregnancy.
ASSAY FaII-E16
This section describes the amplification and detection extension primers for the FaII-E16 Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Table 16, below.

























Amplification Primers*:










FaII-F18
(SEQ ID NO: 13)









5′-ACGTTGGATGACTCATATTCTGGGCTCCTG-3′



 










FaII-R19
(SEQ ID NO: 14)









5′-ACGTTGGATGAGAGAGCTGCCCATGAATAG-3′






Detection Extension Primer:










FaII-E16
(SEQ ID NO: 15)









5′-CACTGGGAGCATTGAGGCT-3′






Termination mix: ddA/ddC/ddG

    
    
        * amplification primers have the 10-mer tag specific for the hME method:acgttggatg.
    
    









TABLE 16







Sequence, length and mass of extension primer,


extended primer (G and A) and pause product)










Allele
Sequence
Length
Mass





FaII-E16
CACTGGGAGCATTGAGGCT
19 bp
5868.8


(G20210A)





 


Negative
CACTGGGAGCATTGAGGCTC
20 bp
6142  


(wild type)





 


Homozygouse
CACTGGGAGCATTGAGGCTTG
21 bp
6486.2


(mutant)





 


Contaminant


6173  


(pausing peak)





(SEQ ID NOS: 825 826, and 827)






FaII-E15 ASSAY

This section describes the amplification and detection extension primers for the FaII-E15 Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Table 17, below:

























Amplification Primers*:










FaII-F10
(SEQ ID NO: 16)









5′-ACGTTGGATGTGGAACCAATCCCGTGAAAG-3′



 










FaII-R11
(SEQ ID NO: 17)









5′-ACGTTGGATGAGAGAGCTGCCCATGAATAG-3′






Amplicon length: 112 bp

    
    
        * amplification primers have the 10-mer tag specific for the hME method: acgttggatg.
    
    


Detection Extension Primer:










FaII-E15
(SEQ ID NO: 18)









5′-CAATAAAAGTGACTCTCAGC-3′






Termination mix: ddA/ddC/ddT







TABLE 17







Sequence, length and mass of extension primer,


extended primer (G and A) and pause product)










Allele
Sequence
Length
Mass





FaII-E15
CAATAAAAGTGACTCTCAGC
20 bp
6094.00


(G2020A)





 


Negative
CAATAAAAGTGACTCTCAGCGA
22 bp
6720.4 


(wild type)





 


Homozygouse
CAATAAAAGTGACTCTCAGCA
21 bp
6391.2 


(mutant)





 


Contaminant


6423.20


(pausing





peak)





(SEQ ID NOS: 830, 831, and 832)






Testing Procedure

Blood specimens and quality control procedures were performed according to protocols described in Example 1. Table 18, below, provides a list of controls used in the presently described assay.







TABLE 18





Controls used in Coagulation Factor II assay



















64Fall030106
A
Factor II G20210A homozygous mutant



NA16000
A
Factor II G20210A homozygous mutant



7740
G
Factor II G20210A negative- wild type



7776
G
Factor II G20210A negative- wild type



GM14899
G
Factor II G20210A negative- wild type



GM7776
G
Factor II G20210A negative- wild type



GM7798
G
Factor II G20210A negative- wild type



GM8810
G
Factor II G20210A negative- wild type



GM9729
G
Factor II G20210A negative- wild type



NA14646
G
Factor II G20210A negative- wild type



NA14650
G
Factor II G20210A negative- wild type



NA14899
G
Factor II G20210A negative- wild type



NA16028
AG
Factor II G20210A heterozygous










PCR, dephosphorylation, homogenous MASSEXTEND (HE,and spectroclean resin clean up procedures were carried out as described in Example 1, above.
Results Analysis
Results were analyzed as described in Example 1, above. FIG. 4 is a mass spectrum of a heterozygous “GA” allele (heterozygous positive) generated using a screen for a G20210A mutation in the Coagulation Factor II (FII) gene. Exemplary test results are provided in Tables 19 and 20, below.







TABLE 19







FaII-E15















Low


Source



Probability


of DNA
Conservative
Moderate
Aggressive
(No results)














ARUP DNAs
126
4
0
2


Coriell
8
0
0
0


control DNAs









Results of this assay were in 100% in agreement with results from ARUP. Negative controls (homozygous wild type) GM7798, GM8810, GM9729, GM14899, NA14646, NA14650 (Coriell Institute) were homozygous GG, and positive heterozygous control NA 16028 (Coriell) was AG, and positive homozygous mutant control NA16000 (Coriell Institute) was AA. Due to blood clogging the DNA was not extracted and could not be re-extracted because of the too small volume of blood obtained (<0.5 ml). Two samples that had no DNA gave no signal. As indicated in Table 19, out of 140 samples tested, the majority of geneotype calls for this assay were conservative calls, indicating a high level of accuracy.







TABLE 20







FaII-E16















Low


Source



Probability


of DNA
Conservative
Moderate
Aggressive
(No results)














ARUP DNAs
128
0
1
3


Coriell
8
0
0
0


control DNAs









Results of this assay were in 100% in agreement with results from ARUP. Negative controls (homozygous wild type) GM7798, GM8810, GM9729, GM14899, NA14646, NA14650 (Coriell Institute) were homozygous GG, and positive heterozygous control NA16028 (Coriell) was AG, and positive homozygous mutant control NA16000 (Coriell Institute) was AA. Due to blood clogging the DNA was not extracted and could not be re-extracted because of the too small volume of blood obtained (<0.5 ml). Two samples that had no DNA gave no signal. As indicated in Table 20, out of 140 samples tested, the majority of geneotype calls for this assay were conservative calls, indicating a high level of accuracy.
Example 3
Screen for Coagulation Factor V R506Q Mutations
The Factor V mutation (Factor V Leiden) is the most common genetic cause of venous thrombosis. It is involved in 20-40% of cases and is present in 3% of the general population. In a population study of 180 individuals from Germany, the heterozygosity rate was 7.8% with a confidence interval 4 to 11%. The mutation causes resistance to activated protein C (APC) and induces a defect in the natural anticoagulation system. The other major genetic causes of venous thrombosis (deficiencies of protein C, protein S and antithrombin III) together account for only 5-10% of cases.
Presence of the factor V mutation increases the risk for venous thrombosis 7-fold in heterozygotes and 80-fold in homozygotes. This risk is increased still further when the factor V mutation is present along with situations such as pregnancy, oral contraceptive use, estrogen therapy, malignancy, diabetes mellitus, immobilization or surgery. Ten percent of heterozygotes and almost all homozygotes experience venous thrombosis in their lifetime. The discovery of the factor V mutation has revolutionized the diagnostic work-up of patients with hypercoagulability, and the ability to detect this mutation in asymptomatic relatives offers the opportunity to prevent venous thrombosis through special management of those at risk.
Gene Information
Gene Locus: 1q23
Gene Symbol: F5 (FV)
Gene Product: Coagulation Factor V
Mutations: 1691 G-A; R506Q; Factor V Leiden
Mutation Frequency: 2-8% in most populations
OMIM number: 227400
mRNA NCBI Acc No.: XM 171908, XM 118651, XM 069970
Genomic Acc No.: NM—000130
Clinical Significance
Testing can be recommended, for example, in patients: having venous thrombosis or pulmonary embolism, transient ischemic attacks or premature stroke, peripheral vascular disease, particularly lower extremity occlusive disease, a history of a thrombotic event, family history of thrombosis or known factor V mutation in a relative; prior to major surgery, pregnancy, postpartum, oral contraceptive use or estrogen therapy if there is a personal or family history of thrombosis; or where there has been a previous finding of activated protein C resistance by laboratory analysis.
FaV-E10 Assay
This section describes the amplification and detection extension primers for the FaV-E10 Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Table 21, below.

























Amplification Primers*:










FaV-F16:
(SEQ ID NO: 19)









5′-acgttggatgAAGACCATACTACAGTGACG-3′



 










FaV-R17:
(SEQ ID NO: 20)









5′-acgttggatgCATTATTTAGCCAGGAGACC-3′






Extension Primer:










FaV-E10:
(SEQ ID NO: 21)









5′-GACAAAATACCTGTATTCCT-3′






    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Amplicon length: 189 bp
Termination mix: ddA/ddC/ddG







TABLE 21







Sequence, length and mass of extension primer,


extended primer (C and T) and pause product










Allele
Sequence
Length
Mass





FaV-E10 R506Q
GACAAAATACCTGTATTCCT
20 bp
6060  


 


C (R) wild
GACAAAATACCTGTATTCCTC
21 bp
6333.2


type





 


T (Q) mutant
GACAAAATACCTGTATTCCTTG
22 bp
6677.4


 


Contaminant


6364.2


(pausing peak)





(SEQ ID NOS: 835, 836, and 837)






FaV-E9 Assay

This section describes the amplification and detection extension primers for the FaV-E9 Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Table 22, below.

























Amplification Primers*:










FaV-F18:
(SEQ ID NO: 22)









5′-acgttggatgCTCTGGGCTAATAGGACTAC-3′



 










FaV-R19:
(SEQ ID NO: 23)









5′-acgttggatgCTGAAAGGTTACTTCAAGGAC-3′






Detection Extension Primers:










FaV-E9:
(SEQ ID NO: 24)









5′-GCAGATCCCTGGACAGGC-3′






    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Amplicon length: 92 bp
Termination mix: ddA/ddC/ddT







TABLE 22







Sequence, length and mass of extension primer,


extended primer (C and T) and pause product










Allele
Sequence
Length
Mass





FaV-E9 R506Q
GCAGATCCCTGGACAGGC
18 bp
5509.6


 


C (R) wild type
GCAGATCCCTGGACAGGCGA
20 bp
6136  


 


T (Q) mutant
GCAGATCCCTGGACAGGCA
19 bp
5806.8


 


Contaminant


5838.8


(pausing peak)





(SEQ ID NOS: 840, 841, and 842)






Testing Procedure

Blood specimens and quality control procedures were performed according to protocols described in Example 1. Table 23, below, provides a list of controls used in the presently described assay.







TABLE 23





Controls used in Coagulation Factor V assay




















11313817
T
Factor V R506Q
homozygous mutant



3109017
T
Factor V R506Q
homozygous mutant



GM10701
C
Factor V R506Q
negative- wild type



GM11053
C
Factor V R506Q
negative- wild type



GM12784
C
Factor V R506Q
negative- wild type



GM14646
C
Factor V R506Q
negative- wild type



NA14646
C
Factor V R506Q
negative- wild type



GM9730
C
Factor V R506Q
negative- wild type



GM8811
C
Factor V R506Q
negative- wild type



GM8914
CT
Factor V R506Q
heterozygous



GM8915
CT
Factor V R506Q
heterozygous



NA14641
CT
Factor V R506Q
heterozygous



NA14650
CT
Factor V R506Q
heterozygous



NA16028
CT
Factor V R506Q
heterozygous










PCR, dephosphorylation, homogenous MASSEXTEND (HME), and spectroclean resin clean up procedures were carried out as described in Example 1, above.
Results Analysis
Results were analyzed as described in Example 1, above. FIG. 5 is a mass spectrum of a heterozygous “GA” allele (heterozygous positive) generated using the FaV-E10 screen for a mutation in the Coagulation Factor V gene. FIG. 6 is a mass spectrum of a heterozygous “GA” allele (heterozygous positive) generated using the FaV-E9 screen for a mutation in the Coagulation Factor V gene. Exemplary test results are provided in Tables 24 and 25, below.







TABLE 24







FaV-E9















Low



Conserv-
Mod-

Probability


Source of DNA
ative
erate
Aggressive
No Results














68 ARUP DNAs
65
2
0
1


71 ARUP DNAs
66
2
0
3


 8 Coriell DNAs
8
0
0
0


 2 UMMHC
2
0
0
0


Total # of DNAs 149
140
5
0
4
















TABLE 25







FaV-E10















Low



Conserv-
Mod-

Probability


Source of DNA
ative
erate
Aggressive
No Results














68 ARUP DNAs
65
2
0
1


71 ARUP DNAs
66
2
0
3


 8 Coriell DNAs
8
0
0
0


 2 UMMHC
2
0
0
0


Total # of DNAs 149
140
5
0
4









Due to blood clogging the DNA was not extracted and could not be re-extracted because of the too small volume of blood obtained (<0.5 ml). As indicated in Tables 24 and 25, above, the majority of geneotype calls for each assay were conservative calls, indicating a high level of accuracy.
Example 4
Cytomegalovirus (CMV) Testing
Clinical Significance
CMV is an opportunistic infection found throughout all geographic locations and socioeconomic groups, infecting about 50% of the general population of the United States by 40 years of age and 90% of people with HIV. CMV is transmitted from person to person by close personal contact. Infectious CMV may be shed in the bodily fluids of any previously infected person, and thus may be found in urine, saliva, blood, tears, semen, and breast milk. CMV can be sexually transmitted and can also be transmitted via breast milk, transplanted organs, and rarely from blood transfusions. In most healthy people, CMV remains in the body indefinitely without causing any harm. Therefore, CMV infection may not be a serious problem for the vast majority of infected individuals.
However, for those with a weakened immune system, including those infected with HIV, or who have undergone bone marrow transplantation or chemotherapy related to cancer, a CMV infection can cause serious and possibly fatal complications. Furthermore, generalized infection and infections of the central nervous system may occur and can be fatal. CMV is also frequently transmitted to a developing child before birth. CMV infection is widespread in developing countries and in areas of lower socioeconomic conditions. For most healthy individuals who acquire CMV after birth, there are few symptoms and no long-term health consequences. Some individuals with symptoms experience a mononucleosis-like syndrome with prolonged fever and a mild hepatitis. Once an individual becomes infected, the virus remains within that individual's body for life, often in a dormant state. Recurrent disease rarely occurs unless the individual's immune system is suppressed, e.g., due to therapeutic drugs or disease.
Infection with CMV is a major cause of disease and death in immunocompromised patients, including organ transplant recipients, patients undergoing hemodialysis, patients with cancer, patients receiving immunosuppressive drugs, and HIV-infected patients. Pneumonia, retinitis (an infection of the eyes), and gastrointestinal disease are the common manifestations of disease.
Because of this risk, exposing immunosuppressed patients to outside sources of CMV should be minimized. Whenever possible, patients without CMV infection should be given organs and/or blood products that are free of the virus.
The risk of developing CMV disease in patients is directly related to the presence and quantity of CMV DNA in blood. The ability to measure CMV viral load is a valuable tool in the management of HIV-1 infected patients. The infection is treatable with drugs. Optimum treatment depends on early and accurate detection.
Overview of Assay
A quantitative CMV assay combines competitive PCR with MassEXTENDTM procedures and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Following isolation of DNA from plasma, PCR amplification is performed on CMV DNA mixed with a known amount of internal standard (oligo—synthetic DNA molecule), which resembles the sequence of the targeted CMV DNA region in all positions except a single base which serves as an assay target. MassARRAY Typer Gene Expression AnalysisTM software allows calculating the ratio of internal standard and patient CMV on each spot and the viral concentration in the extracted sample, which is further recalculated into viral particles per ml of patient sample. The method can be used for sensitive and accurate detection of viral nucleic acid with a broad dynamic range (e.g., from 100 to 4×108 viral copies/ml).
The CMV560 quantitative assay quantifies CMV DNA extracted from plasma of CMV infected individuals using PCR and mass spectrometry. The test can quantify CMV DNA over the range of 100-400,000,000 CMV copies/ml.
The assay can be used in conjunction with clinical presentation and other laboratory markers of disease status as an aid to management of individuals infected with CMV. CMV quantitative results from the assay can be used to assess prognosis of disease progression and to monitor the efficacy of antiretroviral therapy by measuring changes in CMV levels during the course of therapy.
This example generally describes amplification and detection extension primers for detecting the presence and amount of CMV in a biological sample. The sequences of the primers are provided below. The procedures described in Example 1, above, can be modified as necessary for handling and analyzing biological samples containing viruses.
460 E3 Assay

























Amplification Primers*:










460 F1
(SEQ ID NO: 25)









5′-ACGTTGGATGGTCGTGTATGCCACTTTGAC-3′



 










460 R2
(SEQ ID NO: 26)









5′-ACGTTGGATGTGAGGCTGTAATCGCACAGC-3′






Detection Extension Primer:










460 E3
(SEQ ID NO: 27)









5′-CCACTTTGACATTACACCC-3′






    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Termination mix: ddA/ddC/ddT
The sequences of the detection extension primers as they would exist after the extension reaction are provided in Table 26, below.







TABLE 26







Sequence, length and mass of extension primer











Allele
Sequence
Mass






460 E3
CCACTTTGACATTACACCC
5667.7 Da


 



variant A
CCACTTTGACATTACACCCA
5964.9 Da


 



variant G
CCACTTTGACATTACACCCGT
6285.1 Da





(SEQ ID NOS: 845, 846, and 847)






520 E10 Assay


























Amplification Primers*:










520 F8
(SEQ ID NO: 28)









5′-ACGTTGGATGTCTTTCAGGAGACGGGTACG-3′



 










520 R9
(SEQ ID NO: 29)









5′-ACGTTGGATGAGATGAGCAGCTTCTGCAGC-3′






Detection Extension Primer:










520 E10:
(SEQ ID NO: 30)









5′-TCTGCGCGAATGTTACCA-3′






    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Termination mix: ddA/ddC/ddT
The sequences of the detection extension primers as they would exist after the extension reaction are provided in Table 27, below.







TABLE 27







Sequence, length and mass of extension primer











Allele
Sequence
Mass






520 E10:
TCTGCGCGAATGTTACCA
5474.6 Da


 



Variant C:
TCTGCGCGAATGTTACCAC
5747.8 Da


 



Variant G:
TCTGCGCGAATGTTACCAGC
6077   Da





(SEQ ID NOS: 850, 851, and 852)






591 E14 Assay















Wild type target




(SEQ ID NO: 853)


 


Mutated target




(SEQ ID NO: 854)









Amplification Primers*:










591 F12
(SEQ ID NO: 31)









5′-ACGTTGGATGAGGCGTTGCTCTTTAAGCAC-3′



 










591 R13
(SEQ ID NO: 32)









5′-ACGTTGGATGAGTGCGTGAGCTTACCGTTC-3′






Detection Extension Primer:










591 E14
(SEQ ID NO: 33)









5′-TTAAGCACGCCGGCGCGG-3′






    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Termination mix: ddA/ddC/ddG
The sequences of the detection extension primers as they would exist after the extension reaction are provided in Table 28, below.







TABLE 28







Sequence, length and mass of extension primer











Allele
Sequence
Mass






591 E14
TTAAGCACGCCGGCGCGG
5525.6 Da


 



Variant C:
TTAAGCACGCCGGCGCGGC
5798.8 Da


 



Variant T:
TTAAGCACGCCGGCGCGGTC
6103   Da





(SEQ ID NOS: 855, 856, and 857)






607 E30 Assay















Wild type target




(SEQ ID NO: 858)


 


Mutated target




(SEQ ID NO: 859)









Amplification Primers*:










607 F28
(SEQ ID NO: 34)









5′-ACGTTGGATGTCATTTGCGCCGCCAGAATG-3′



 










607 R29
(SEQ ID NO: 35)









5′-ACGTTGGATGTTGCTCTTTAAGCACGCCGG-3′






Detection Extension Primer:










607 E30
(SEQ ID NO: 36)









5′-GCCGCCAGAATGAGCAGA-3′






    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Termination mix: ddA/ddC/ddG
The sequences of the detection extension primers as they would exist after the extension reaction are provided in Table 29, below.







TABLE 29







Sequence, length and mass of extension primer











Allele
Sequence
Mass






607 E30
GCCGCCAGAATGAGCAGA
5542.6 Da


 



Variant A:
GCCGCCAGAATGAGCAGATA
6144   Da


 



Variant G:
GCCGCCAGAATGAGCAGAC
5815.8 Da





(SEQ ID NOS: 860, 861, and 862)






CV33-E Assay















Wild type target




(SEQ ID NO: 863)


 


Mutated target




(SEQ ID NO: 864)









Amplification Primers*:










CV-F31
(SEQ ID NO: 37)









5′-ACGTTGGATGGAGACGACGTGGACGGCAC-3′



 










CV-R32
(SEQ ID NO: 38)









5′-ACGTTGGATGCGTGCTTGGACACGCGACTT-3′






Detection Extension Primer:










CV33 E
(SEQ ID NO: 39)









5′-CTCCTTGTCCGAAGCCGC-3′






    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Termination mix: ddA/ddC/ddT
The sequences of the detection extension primers as they would exist after the extension reaction are provided in Table 30, below.







TABLE 30







Sequence, length and mass of extension primer











Allele
Sequence
Mass






CV33 E
CTCCTTGTCCGAAGCCGC
5411.5 Da


 



Variant C:
CTCCTTGTCCGAAGCCGCGT
6028.9 Da


 



Variant T:
CTCCTTGTCCGAAGCCGCA
5708.7 Da





(SEQ ID NOS: 865, 866, and 867)






CMV 560 Assay

For quantitative PCR the region chosen is situated in the human CMV UL97 region that codes for a viral serine/threonine protein kinase. A standardized quantitation panel was prepared with CMV strain AD169. The CMV stock of 4×1010 was diluted with human negative plasma (Accrometrix) to concentrations of 40 copies/ml to 4×108 CMV copies per mililiter.
FIGS. 13A1-13G3 illustrate typical results of the CMV assay. FIGS. 13A1-13A3 are a set of mass spectra in a CMV quantitative assay on samples containing 400 CMV copies/ml compared to internal controls. FIGS. 13B1-13B3 are a set of mass spectra in a CMV quantitative assay on samples containing 4000 CMV copies/ml compared to internal controls. FIGS. 13C1-13C3 is a set of mass spectra in a CMV quantitative assay on samples containing 40,000 CMV copies/ml compared to internal controls. FIGS. 13D-13D3 is a set of mass spectra in a CMV quantitative assay on samples containing 400,000 CMV copies/ml compared to internal controls. FIGS. 13E1-13E3 are a set of mass spectra in a CMV quantitative assay on samples containing 4,000,000 CMV copies/ml compared to internal controls. FIGS. 13F1-13F3 is a set of mass spectra in a CMV quantitative assay on samples containing 40,000,000 CMV copies/ml compared to internal controls. FIGS. 13G1-13G3 is a set of mass spectra in a CMV quantitative assay on samples containing 400,000,000 CMV copies/ml compared to internal controls.














Wild type CMV template




(SEQ ID NO: 868)


 


Mutated oligo template Extension primer CMV560




(SEQ ID No: 869)









Amplification Primers*:










CMV560-S:
(SEQ ID NO: 58)









5′-ACGTTGGATGCGCTTCTACCACGAATGCTC-3′



 










CMV560-L:
(SEQ ID NO: 59)









5′-ACGTTGGATGATAAATACAGCCCGTCGCTC-3′






Detection Extension Primer:










CMV560-E
(SEQ. ID NO: 60)









5′-CTTTCTGACGTATTCGTGCAGCAT-3′






    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Amplicon length: 94 bp
Termination mix: ddA/ddC/ddT
The sequences of the detection extension primers as they would exist after the extension reaction are provided in Table 31, below.







TABLE 31







Sequence, length and mass of extension primer










Allele
Sequence
Length
Mass





CMV560Quant
CTTTCTGACGTATTCGTGCAGCAT
24 bp
7309.8


 


C wild type
CTTTCTGACGTATTCGTGCAGCATG
27 bp
8256.4


 



GT




 


T mutant
CTTTCTGACGTATTCGTGCAGCATA
25 bp
7607  


 


Contaminant


7639  


(1st pause)





(SEQ ID NOS: 870, 871, and 872)






Testing Procedure

This section generally describes exemplary procedures used in CMV assays.

Type of Specimen
Blood was collected observing universal precautions for venipuncture.
For plasma samples, blood was collected in sterile tubes containing EDTA (K2) or ACD, and stored at room temperature. Plasma was removed from cells within 4 hours of collection. The samples were separated by centrifugation at 1000×g for 10 to 15 minutes and standard laboratory procedures were used to remove the plasma. Specimens were not clarified by filtration or further centrifugation.
Specimen Storage and Stability
Specimens were stored at −60° to −80° C. in sterile, screw-capped tubes. Specimens may also be stored at 2° to 8° C. for up to 48 hours or at −20° C. in a non-frost free freezer for up to 72 hours prior to freezing at −60° to −80° C. Repeated freeze/thaw of samples was avoided. Specimens were shipped frozen on dry ice and packaged and labeled in compliance with federal and international regulations covering the transport of clinical samples and etiologicalagents.
Sample Volume
This assay was performed with 500 μl of sample for a single determination. At least 1 ml of plasma was used in the tube.
Processing Whole Blood
Specimens were centrifuged at 1000×g for 10-15 minutes within 4 hours of blood collection. Plasma was aseptically removed and aliquoted (without disturbing the white cell layer or clot) into sterile unbreakable tubes (duplicate aliquots of at least 1 mL plasma are advised to avoid freeze-thaw of specimen in case of repeat testing). Plasma was immediately frozen at −60° C. to −80° C. within 30 minutes of separation. Specimens can be stored at −20° C. in a non-frost-free freezer for up to 72 hours.
Criteria for Unacceptable Specimens
Volume of plasma was below 1 ml.
Barcode of the tube was missing or not readable.
Inappropriate specimens, e.g., blood instead of plasma.
Specimen as received was already defrosted.
Specimen Handling
Blood samples were received with the lab barcode identification numbers. The sample identification was ensured throught out the process by the tracking system on FreedomTM, BioRobot MDxTM, GenesisTM, and MasARRAYTM systems. Specimens were handled as if capable of transmitting infection. Specimen was stored immediately after receipt in the −80 C freezer were not processed immediately.
Materials and Equipment
I. Reagents used for PCR amplification: 10 mM dNTPs (Invitrogen), 10 μM primer mix (forward and reverse) (IDT), internal control—oligo (IDT), PCR kit (Qiagen), sterile water, 10% bleach. Each kit (Qiagen) contains the following reagents: 10×PCR/MgCl2 buffer; 25 mM MgCl2; and HotStar Taq DNA Polymerase.
Volumes that were used for the PCR reactions are provided in Table 32, below.







TABLE 32







Composition of PCR reaction













Reaction


Reagent
Stock Concentration
Final Concentration
Volume














Water
N/A
N/A
0.28
μl


Buffer/MgCl2
15 mM (10×)
1.5 mM
2.0
μl


MgCl2
25 mM (10×)
2.5 mM
0.8
μl


dNTPs
10 mM (50×)
200 μM
0.16
μl


Primer Mix
10 μM (×)
200 nM
0.4
μl


Internal std
Variable (see below)
Variable (see below)
0.2
μl


CMV560 C_T






HotStarTaq
5 units/μl (50×)
0.1 unit per reaction
0.16
μl


TOTAL volume


4.00
μl









Reagent Preparation
Oligo-standard (internal control or internal standard) stock solutions were made. The oligo-standard comes in dry-form. The 20 μM solution was made using the following equation for calculating the volume of water needed: μl water=nmol of oligo×1000 μl/20 nmol. The 20 μM solution of the oligo-standard oligo-standard was thawed and vortexed before use if frozen. A 1 μM stock of oligo-standard was made by diluting 20 μl of 20 μM oligo-standard solution in 380 μl of a 1 ng/μl carrier DNA. A series of working solutions was made in 10E-10M to 10E-20M in 10-fold increments by diluting the Oligo Standard (internal control) 1 μM stock solution using the Tecan Gemini program Diluting oligos10x.gem on C:\Gemini\Data\CMV. 10× dilutions were made in 1 ng/μl carrier DNA.
Master mix was prepared with primers as follows: the 10×PCR Buffer, dNTP mix, 25 MM MgCl2, and stock primers were thawed and vortexed before use. The solutions were mixed completely before use to avoid localized salt concentrations. The PCR mixes w/primers were made according to Table 32, above, multiplying the reagent volumes by number of samples and factor which depends on number of samples run.
II. Reagents used for MassARRAYTM: SpectroCHiP, 384-well microplates for PCR (Marsh #SP0401Sequen); 96-well polypropylene skirted microplates for resin addition (Marsh #AB-0800); 96-well polystyrene v-bottom microplates for use with liquid handler (Sarstedt #82.1583); Adhesive PCR foil seals for freezing plates (Marsh #AB-0626); Adhesive Plate seals for temporary sealing (Marsh #AB-0580); Adhesive PCR seals for use in thermocycling (Marsh #AB-0558 or ABI MicroAmp #4306311); 20 ul tips for the liquid handler (VWR #BK717254); tips for manual pipettors; tubes for mixing reagents
III. Other Materials: hME buffer; hME SAP enzyme (Shrimp Alkaline Phosphatase); ACT MassEXTENDTM Mix; ThermoSequenaseTM enzyme; SpectroCLEANTM resin; 3 point calibrant; Autoclaved or sterile water >18.2 MegaOhm/cm resistivity (store in plastic); Deionized >18.2 MegaOhm/cm resistivity water for instrument wash stations; isopropanol for liquid handler cleaning; 50% ethanol for nanodispenser operation (stored in sealed, plastic container); oligonucleotide primers; Desiccant (silica gel with moisture indicator).
IV. Exemplary Hardware/Software
MassARRAYTM Liquid handler (Beckman Multimek) automated 96-channel pipettor with controller PC; MassARRAYTM Nanodispenser (RoboDesign pin tool) micro-arrayer with controller PC; 384-well microplate Thermocycler; MassARRAYTM Genotype Analyzer (Bruker Autoflex MALDI-TOF Mass Spectrometer controlled by SpectroACQUIRE software); MassARRAYTM RT Workstation; MassARRAYTM Assay Design Software; MassARRAYTM Oracle Database; Plate centrifuge; universal plate holders; SpectroCLEANTM resin plate, spoon and scraper; dessiccator for chip storage; single and multi-channel pipettors; plate rotator for mixing resin after a plate has been stored frozen.
Reagent Preparation
Appropriate buffer, primers and EXTENDTM mix were defrosted and vortexed to ensure suspension before using, except for enzymes which were mixed by inversion or pipetting. Enzymes were kept in the freezer or cooler at all times. As enzymes are in low viscosity buffer, they were pipetted slowly to make sure correct volume was applied. Autoclaved Type II water was used to make up solutions.
Reagent Volumes for Sap and Reactions
Dephosphorylation
Shrimp Alkaline Phosphatase (SAP) was prepared in a tube according to Table 33, multiplying the volumes by the number of the samples and then by 1.5 (volumes were for a 384-well microplate and included a 50% overage to account for possible pipetting loss and dead volume in the 96-well microtiter plate used on the Multimek).
Volumes that can be used for the SAP reactions are provided in Table 33, below.







TABLE 33







Composition of SAP mix













Final






Concentration

Volume per



Stock
(with 5 ul
Reaction
384-well


Reagent
Concentration
PCR product)
Volume
plate















Nanopure
N/A
N/A
1.53 μl
881.3
μl


Autoclaved







Water







hME buffer
10×
1×
0.17 μl
97.9
μl


SAP
1 unit/μl
0.04 units/μl
0.30 μl
172.8
μl


Totals:


2.00 μl
1152.00
μl









2 μl of the diluted SAP was dispensed to each well (see testing procedure).
Homogeneous MassExtend (hME) Reaction
hME cocktail was prepared in a tube according to Table 34, below. One reaction volume was multiplied by the number of samples and then by 1.38 (volumes were for a 384-well microplate and included a 38% overage to account for possible pipetting loss and dead volume in the 96-well microtiter plate used on a Multimek).
Volumes that can be used for the hME reactions are provided in Table 34, below.







TABLE 34







Components of hME cocktail













Final






Concentration






(with 7 ul

Volume per



Stock
PCR/SAP volume
Reaction
384-well


Reagent
Conc.
in well)
Volume
plate*















Nanopure
N/A
N/A
1.58 μl
837.3
μl


Autoclaved







Water







hME Mix
10×
1× buffer
 0.2 μl
106.0
μl


(10 × buffer

(together with





w/2.25 mM

PCR buffer) with





d/ddNTPs)

50 uM d/ddNTPs







each





Extension
50 μM
600 nM
0.18 μl
95.4
μl


primer







Thermo
32 units/μl
0.063 unit/μl
0.04 μl
21.2
μl


Sequenase&







Totals:


 2.0 μl
1059.9
μl





&ThermoSequenase was kept at −20° C. until added to the reaction cocktail.






2 μl of the hME cocktail mix was dispensed to the appropriate wells containing 7 μl dephosphorylated PCR product (see Testing Procedure).
Spectroclean Resin Clean Up
Using the SpectroCLEANTM plate, resin was transferred into the 96-well Marsh plate. The SpectroCLEANTM plate controls the volume of resin added.
Acceptable Reagent Performance
The acceptability of a reagent was determined by ensuring that quality control results (negative, high, and low controls) were within acceptance criteria ±0.5 log10 value when received from the manufacturer (Affimetrix).
Reagent Storage and Stability
hME buffer, hME Mix (10× buffer w/d/ddNTPs), SAP enzyme and ThermoSequenaseTM enzyme, calibrant and oligonucleotide primers were stored at −20° C. (tube of calibrant in use was stored at 4° C.). SpectroCLEANTM resin, water and alcohols were stored at room temperature. The resin was stored away from direct sunlight. SpectroCHIPSTM were stored dessicated.
Calibration
Calibration of the Multimek and Autoflex was performed as daily maintenance. Calibration of the SpectroPOINTTM was performed as monthly maintenance. Calibration of the ABI Thermocycler was performed as yearly maintenance. The high, low and negative external standards were run with appropriate internal standards with each run to ensure that oligo-standards were at appropriate concentrations. The eight internal standards (mutated oligos) were run with each patient sample (eight wells).
Standards
The high, low and negative external standards 0/500/5,000/50,000 copies/ml were made from human CMV purified virus, strain AD169 from Advanced Biotechnologies Inc., diluted in human CMV negative plasma (Acrometrix). Standards were diluted from the stock 4×1010 c/ml when needed, assayed concurrently with the previous batch before being used for the quantification of patient specimens. Diluted standards were stored in −80° C. Alternatively, CMV DNA 4 member panel: 0/500/5,000/50,000 copies/ml from Acrometrix (catalog number 94-2014) was used. Pre-assigned values for internal standards were: 1 pm, 100 fM, 10 fM, 1 fM, 100 aM, 10 aM, 1 aM. Standards were assayed with each run according to the plate 384 well map.
Interpreting Results
Procedures Involved In Assays
DNA extraction and concentration measurement, Uniplex PCR Procedure on Genesis with internal standard, and Homogeneous MassEXTEND Reaction using the MassARRAY System were performed using standard procedures.
Quality Control
To monitor assay performance, at least three levels of control material were included with every assay. The assay was considered valid if all the following conditions occurred: the values determined for the CMV positive controls were within ±0.5 log10 specified range and the CMV negative control did not give a positive result. If any of the above conditions are not met: patient results from the affected run should not be reported, the cause for quality control failure should be determined, and the run should be repeated if needed. If the assay must be repeated, then do the following: review instructions to ensure that the assay is performed according to the procedure; verify that the standards and controls are in the appropriate location specified by the plate map; verify that the materials are not expired; verify that the liquid handling robotics are pipetting correct volumes (should be done before the original run).
Controls
All controls were diluted in HIV/HCV/CMV negative human plasma. Pre-assigned values for controls appeared on the tubes: a) The negative control was non-reactive for CMV; b) the positive controls contained a specific CMV titer determined previously or stated by manufacturer (Acrometrix). All controls were be assayed in every plate run according to the order on the tray map. The performance of each control are discussed in the Results section.
Testing Procedures
I. CMV DNA Extraction
CMV DNA was extracted on MDX, Qiagen according to standard procedures. The external standards (240 μl) were diluted and pipetted to S-block under the biological hood.
II. PCR Procedure
The composition of a typical PCR reaction is shown in Table 35, below.







TABLE 35







Composition of single PCR reaction in 384-well plate











Stock
Final
Reaction


Reagent
Concentration
Concentration
Volume





PCR mix w/primers
10×
1×
 4.0 μl


& oligo-standard





CMV DNA
N/A
N/A
16.0 μl


TOTAL volume


20.00 μl









To calculate the volumes per run, the volumes of reagents per reaction in Table 32 were multiplied by the number of reactions and by multiplication factor which depended on the sample number run with each oligo-standard concentration (see Table 36). Increasing the final volume allowed for pipetting losses and ‘dead’ volume when using robotics. The mix was stored at 4° C. until use.







TABLE 36







Multiplication factor in calculations reagent volumes


for PCR reaction using Tecan AssayTransfer.gem


program (8 wells of the same assay mix)












Number of Wells w/the





same Oligo-Standard
Multiplication factor
















1
56




2
28




3
19




4-6
14




7
8




8-9
7




10-11
6




12-13
5




14-18
4




19-22
3




23-26
2.5




27-37
2




>37
1.5










4 μl of PCR mix were dispensed into each well of the 384-well plate. 16 μl of extracted CMV DNA was added to each well using TEMO (Tecan). The following PCR program was run on the thermocycler:

















95° C.
15 minutes





95° C.
20 seconds





56° C.
30 seconds
 {close oversize brace} 
65 cycles



72° C.
 1 minute





72° C.
 3 minutes





 4° C.
forever (hold)









III. Dephosphorylation Procedure:
Before running SAP procedure, 5 μl of the sample were transferred from each well of the original 20 μl 384-well plate to a new 384-well plate using Multimek (program: “PCR-5 μl to 1 384 plate”). If needed, 3 sister plates can be made this way using program “PCR-5 μl to 3 384 plate.” SAP was diluted according to Table 36, above, multiplying the volumes by the number of the samples and then by 1.5. The composition of a typical SAP mix is provided in Table 37, below.







TABLE 37







Composition of SAP Mix












Stock
Final
Reaction
Volume per


Reagent
Concentration
Concentration
Volume
384-well plate















Nanopure
N/A
N/A
1.53 μl
881.3
μl


Water







hME
10×
1× *
0.17 μl
97.9
μl


buffer







SAP
1 unit/μl
0.15 units/μl
0.30 μl
172.8
μl


Total


2.00 μl
1152.00
μl









2 μl of the diluted SAP were dispensed to each well. The SAP PCR program was run on the thermocycler as follows:















37° C.
20 minutes



85° C.
 5 minutes,



 4° C.
forever (hold)









IV. Homogeneous MassExtend (hME) Reaction Procedure:
hME cocktail was prepared according to Table 37, above. One reaction volume was multiplied by the number of samples and then by 1.38. The composition of a typical hME preparation are provided in Table 38, below.







TABLE 38







Components of hME cocktail















Volume per



Stock
Final
Reaction
384-well


Reagent
Concentration
Concentration
Volume
plate*















Nanopure
N/A
N/A
1.58 μl
837.2
μl


Water







hME Mix
10×
1×
 0.2 μl
106
μl


(10 × buffer







w/2.25 mM







d/ddNTPs)







Extension
50 μM
2.7 μM
0.18 μl
95.4
μl


primer







Thermo
32 units/μl
0.063 unit/μl
0.04 μl
21.2
μl


Sequenase&


 2.0 μl
1059.8
μl


TOTALS:


 2.0 μl
1059.8
μl





*Volumes are for a 384- well microplate and include a 38% overage to account for possible pipetting loss and dead volume in the 96-well microtiter plate used on the Multimek.


&ThermoSequenase TM was kept at −20° C. in a portable cooler or freezer at all times.






2 μl of the hME cocktail mix was dispensed to the appropriate wells containing 7 μl dephosphorylated PCR product. The Extension Reaction program was run on the PCR machine as follows:

















94° C.
2 minutes





94° C.
5 seconds





52° C.
5 seconds
 {close oversize brace} 
99 cycles



72° C.
5 seconds





 4° C.
forever (hold)









V. SpectroCLEAN Resin Clean Up Procedure:
Using a SpectroClean plate, resin was transferred into the 96-well Marsh plate. The hME cation Cleanup method was run on the Biomek.
VI. Dispensing Extended Product on a Chip Using Nanodispenser
Extended product was dispensed on chips according to the manufacturer's instructions.
VII. Reading the Chip on Autoflex.
Chips were read according to the manufacturer's instructions.
The specific issues in quantitative assays were:

    
    
        (1) Sample Group in Plate Editor contained in SamplelD column: name of the samples (barcodes) and standards, and Description column contained the concentrations of the standards as molar integers for example 1 aM, 10 aM, 1 fM etc.
        (2) After the run was finished, “Gene Expression” report was used for calculating the molar concentration (M) of the CMV DNA in extracted sample.
        (3) The molar concentrations needed to be recalculated into copies/ml by macro in excel file or written script.
    
    


Exemplary Test Results
FIGS. 13A-13G are spectra in CMV quantitative assays for samples containing 400 to 4×108 copies/ml. The CMV target was read as “C” allele, and the internal standard (IS) as “T” allele. The areas under the signal peaks are were used by Sequenom Quantification software for quantitation of CMV concentration in the sample. Internal Standards (IS): IS5=10E-11M; IS6=10E-12M; IS7=10E-13M; IS8=10E-14M; IS9=10E-15M; IS10=16E-13M; IS11=10E-17M; IS12=10E-18M; IS13=10E-19M.
The data from the Quantitative Assay was measured as the area under the curve (spectrum) for standards, controls, and patient specimens. The concentration of the unknown samples was calculated based on known standards concentration automatically by the Sequenom software MassArray TyperTM version 3.1.4. The external CMV controls were included in each run to ensure the quality of the results. The lower limit for the assay was 100 copies/mL. Specimens quantitating below this cut-off value can be reported as <100 copies/ml. Specimens with values greater than 1×10E8 copies/mL were above the upper limit of quantitation and can be reported as >1×10E8 copies/ml.
Quantitation of CMV required individual interpretation because of the lack of well-established standard levels and the different rates of progression of the infection to overt disease from the time of first detection in different illness (rapid in bone marrow transplant patients, less rapid in patients with HIV-AIDS). The most consistent marker of progression is a rising viral load in an individual patient, rather than an absolute value.
Standards
External standards were diluted CMV: High—4×10E6, Medium—4×10E4, Low—4×10E2 copies/ml. The original stock with 4×10E10 titer was purchesed from ABI Tech, Inc. Internal standards were diluted oligos containing one mismatch at the site that was assayed. Concentration of the internal standards: 1 pM, 100 fM, 10 fM, 1 fM, 100 aM, 10 aM, 1 aM, 0 .aM, 0.01 aM.
Negative Controls

    
    
        1. The area under the curve of the Negative Control should be less than the corresponding value of the cutoff (100 copies/mL).
        2. The assay should be flagged as “Invalid” if the above condition is not met.
    
    


Positive Controls

    
    
        1. The Positive Controls should quantitate within the range ±0.5 log of the value on the standard label for home made standards or on the package insert for Acrometrix standard.
        2. If the Positive Controls quantitate outside the above range, the assay should be flagged “Invalid”.
    
    


Reporting or Results
Results are reported in CMV copies/ml. Results can be saved into a Laboratory computer system and released to the Meditech System after run approval by a Director or designee through the Interface System.
Limitations
The following quantitation limits are defined for CMV QUANT assay:

    
    
        The Limit of Quantitaion (LoQ) is 100 CMV DNA copies/ml.
        The Upper Quantitation Limit (UQL) for this assay has been determined to be 400,000,000 CMV copies/ml.
        The quantitation range of the assay is from 100 to 400,000,000 CMV copies/ml.
    
    


Multiple Freeze/Thaw Cycles
The effect of 1, 2, 3 and 4 freeze-thaw cycles frozen at −60° to −80° C. was tested on 40 CMV negative and 25 CMV-positive serum specimens. Up to 3 freeze-thaw cycles on CMV-negative or CMV-positive specimens had no effect on the CMV Quant assay performance.
SPECIFICITY of the CMV560 ASSAY—Herpes Virus GROUP
CMV (HSV type 5) is a member of the herpes virus group, which includes herpes simplex virus types 1 and 2, varicella-zoster virus, HSV type 4, (which causes chickenpox), and Epstein-Barr virus, HSV type 3, (which causes infectious mononucleosis). Specificity of the CMV560 assay was determined by testing amplification primers and extend probe against each of the four types, above mention, herpes virus reference standards using 103 DNA copies in each reaction. CMV560 assay was specific only for CMV virus. Also, positive specific tests were run for each of the other type viruses to ensure that there was specific DNA target.
Linearity
The Linearity of the CMV assay extends over seven logs. An aliquot of quantitated CMV strain AD169 (Advanced Biotechnologies, Columbia, Md.) serially diluted with CMV negative human plasma to concentrations of 4×108 to 40 tcopies/ml was used for assessing the linearity of the assay. Sixteen tests were run for each viral concentration in 10-fold increments from 40 to 4×108 copies/ml (4×4 replicates) and 12 tests for viral concentrations of 4×107 and 4×108 copies/ml (3×4 replicates). The linear range of the CMV assay in this experiment was from 400-40,000,000 copies/ml (FIG. 14). The standard deviation of values in this linear range (over 7 log10) was from 0.001-0.29 log10, and viral load values agreed within 2-fold.
Sensitivity of the Assay
To increase sensitivity of the assay, total volume of the reaction was increased from 5 to 20 μl and the volume of CMV DNA in the reaction was increased 8 folds (from 2 to 16 μl). After this change the MassARRAYTM assay reached a sensitivity of 100% (all samples called) when the input target amount was greater than 100 copies/ml.
Example 5
Screen for Hemachromatosis Mutations
Hereditary hemochromatosis is classically inherited as a recessive trait but is genetically heterogenous. Mutations in the HFE and the TFR2 (transferrin receptor2; Y250X) genes account for about 80% of patients, while a third locus on chromosome 1q is responsible for juvenile hemochromatosis. The nonclassical form of iron overload inherited as an autosomal dominant trait is caused by the mutation N144H in the SLC11A3 protein.
Hematochromatosis is a condition that causes the intestine to absorb too much iron. Over time (often several years) this excess iron is deposited in the cells of the liver, heart, pancreas, joints, and pituitary gland. If untreated, organ damage can result. Iron overload can cause, e.g., liver cancer, diabetes, cirrhosis of the liver, heart disease, arthritis, gray or bronze skin pigmentation, impotence, infertility, and amenorrhea.
The age of onset varies, but symptoms generally begin during middle age. Since hemochromatosis is a hereditary disease, the individuals in the family of a diagnosed individual are at higher risk for having gene mutations. Individuals having a mutation(s) should have iron overload screening since affected individuals are at higher risk for developing iron overload.
There are at least five types of hemochromatosis. Juvenile hemochromatosis or hemochromatosis type 2 (HFE2) is an autosomal recessive disorder. One form, which is designated HFE2A, maps to 1q21. A second form, designated HFE2B, is caused by mutation in the gene encoding hepcidin antimicrobial peptide and maps to 19q13. Hemochromatosis type 3 (HFE3) is an autosomal recessive disorder, caused by mutation in the gene encoding transferrin receptor-2 isoform (TFR2), which maps to 7q22. Hemochromatosis type 4 (HFE4), an autosomal dominant disorder, is caused by mutation N144H in the SLC11A3 gene (604653) that maps to 2q32 and encodes ferroportin 1/IREG1/MTP1—an intestinal iron transporter. Hemochromatosis type 5 is autosomal dominant and caused by mutation in H-ferritin—iron responsive element.
Gene Information
Gene Locus: 6p21.3
Gene Symbol: HFE
Gene Product: HFE protein
Mutations: H63D; C282Y; S65C
Mutation Frequency: The prevalence of C282Y homozygosity, H63D homozygosity, and C282Y/H63D compound heterozygosity is estimated to be 0.26%, 1.89% and 1.97%, respectively. The prevalence estimates for C282Y heterozygosity (i.e. C282Y/wild type) are 9.54% among non-Hispanic white population, 2.33% among non-Hispanic blacks, and 2.75% among Mexican-Americans. The prevalence estimates of the C282Y mutation in the US population and of the H63D mutation are 5.4% and 13.5%, respectively.
OMIM number: 235200
mRNA NCBI Acc No.: NM—000410: hemochromatosis mRNA (HFE); U60319: haemochromatosis protein (HLA-H); XM—030153: human hemochromatosis mRNA (HFE)
Genomic Acc No.: NG—001335 Homo sapiens genomic large histone family cluster (HFL@) on chromosome 6 (NCBI); NT—007592 Homo sapiens chromosome 6 genomic contig (NCBI); chr 6|GA_x5L2HTUTQ9V (Celera Database)
Clinical Significance
The test can indicate the presence or absence of three mutations that are associated with hereditary hemochromatosis: CYS282TYR (C282Y), HIS63ASP (H63D), and S65C.
CYS282TYR is a missense mutation caused by a G-to-A change at nucleotide position 845 in exon 4 that results in a cysteine to tyrosine transition at position 282 [Cys282Tyr (C282Y)] in the HFE protein. More than 80% of patients with hereditary hemochromatosis are homozygous for this mutation.
HIS63ASP is a mutation caused by a C-to-G change in exon 2 that results in a histidine to asparagine subsitution at position 63 [His63Asp (H63D)] in the HFE protein. Heterozygotes for this mutation are highly prevalent in the general population. This mutation has been found in higher frequency in patients heterozygous for the C282Y substitution than in control individuals. In the homozygous state, His63Asp has been also been observed in a few patients with hereditary hemochromatosis. It is still unclear, however, whether this mutation is a neutral polymorphism or a mutation that in the presence of the C282Y mutation results in a more affected phenotype.
S65C is a mutation, A193T, resulting in a serine to cysteine change at amino acid 65 (S65C) that appears to be associated with milder forms of hereditary hemochromatosis.
The HFE screens described herein can be recommended for a patient, for example, when a member of the patient's family has been diagnosed with HFE and/or when evidence of iron overload exists.
ASSAY For H63D:
This section describes the amplification and detection extension primers for the H63D Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Tables 31 and 32, below.
HFE H63D (C187G) Mutation in Exon 2:
Assay: FM3-E
Overview:
Reverse Reaction:














Wild type template




(SEQ ID NO: 873)


 


Mutated template




(SEQ ID NO: 874)


 


Extension primer




(SEQ ID NO: 875)









Amplification by PCR*:











HFE-F1
(SEQ ID NO: 40)









5′-ACGTTGGATGATGACCAGCTGTTCGTGTTC-3′



 










HFE-R2
(SEQ ID NO: 41)









5′-ACGTTGGATGTCTACTGGAAACCCATGGAG-3′






Amplicon length: 74 bp

    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Extension:
Terminator Bases Mix: ACT








Extend Primer (FM3-E):
(SEQ ID NO: 42)







5′ CTCCACACGGCGACTCTCAT-3′(reverse primer)


 








Analyte C:
(SEQ ID NO: 876)







5′ CTCCACACGGCGACTCTCATGA


 








Analyte G:
(SEQ ID NO: 877)







5′ CTCCACACGGCGACTCTCATC 3′






Genotype Calls: C: negative (wild type),

    
    
        G: homozygous (mutant),
        GC: heterozygous.
    
    









TABLE 39







Sequence, length and mass of


extension primer, extended primers and pause


product for HFE FM3-E assay, mutation H63D (C187G)










Allele
Sequence
Length
Mass





FM3-E
CTCCACACGGCGACTCTCAT
20 bp
5997.9 Da


 


C (His)
CTCCACACGGCGACTCTCATGA
22 bp
6624.4 Da


NEGATIVE





 


G (Asp)
CTCCACACGGCGACTCTCATC
21 bp
6271.2 Da


HOMOZYGOUS





 


Pause
CTCCACACGGCGACTCTCATG
21 bp
6327.2 Da





(SEQ ID NOS: 878, 879, 880)






Assay: HFE-E3

Overview:














Wild type template




(SEQ ID NO: 881)


 


Mutated template




(SEQ ID NO: 882)


 


Extension primer




(SEQ ID NO: 45)









Amplification by PCR*:










HFE-F7
(SEQ ID NO: 43)









5′-ACGTTGGATGTTCATGGGTGCCTCAGAGC-3′



 










HFE-R8
(SEQ ID NO: 44)









5′-ACGTTGGATGCCACATCTGGCTTGAAATTC-3′






Amplicon length: 153 bp

    
    
        * amplification primers have the 10-mer tag specific for hME method:acgttggatg.
    
    


Extension:
Terminator Bases Mix: ACT










Extend Primer (HFE-E3):
(SEQ ID NO: 45)









5′ CAGCTGTTCGTGTTCTATGAT 3′



 










Analyte C:
(SEQ ID NO: 883)









5′ CAGCTGTTCGTGTTCTATGATC 3′



 










Analyte G:
(SEQ ID NO: 884)









5′ CAGCTGTTCGTGTTCTATGATGA 3′






Genotype Calls: C negative (wild type),

    
    
        G homozygous (mutant),
        GC heterozygous.
    
    









TABLE 40







Sequence, length and mass of


extension primer, extended primers and pause


product for HFE-E3 assay, mutation H63D (C187G)










Allele
Sequence
Length
Mass





HFE-E3
CAGCTGTTCGTGTTCTATGAT
21 bp
6418.2 Da


 


C (His)
CAGCTGTTCGTGTTCTATGATC
22 bp
6691.4 Da


NEGATIVE





 


G (Asp)
CAGCTGTTCGTGTTCTATGATGA
23 bp
7044.6 Da


HOMO-





ZYGOUS





 


Pause
CAGCTGTTCGTGTTCTATGATG
22 bp





(SEQ ID NOS: 885, 886, and 887)






HFE C282Y (G845A) Mutation in Exon 4:

This section describes the amplification and detection extension primers for the C282Y Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Tables 33 and 34, below.
Assay: FM6-E
Overview:
Reverse Reaction:













Wild type template 



(SEQ ID NO: 888)


 


Mutated template 



(SEQ ID NO: 889)


 


Extension primer 



(SEQ ID NO: 890)









Amplification by PCR*:











HFE-F4
(SEQ ID NO: 46)









5′-ACGTTGGATGTGGATAACCTTGGCTGTACC-3′



 










HFE-R5
(SEQ ID NO: 47)









5′-ACGTTGGATGTATCACAATGAGGGGCTGATC-3′






Amplicon length: 89 bp

    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Extension:
Terminator Bases Mix: ACG










Extend Primer (FM6-E):
(SEQ ID NO: 48)









5′ GCCTGGGTGCTCCACCTGG 3′



(reverse primer)



 










Analyte G:
(SEQ ID NO: 891)









5′ GCCTGGGTGCTCCACCTGGC 3′



 










Analyte A:
(SEQ ID NO: 892)









5′ GCCTGGGTGCTCCACCTGGTA 3′






Genotype Calls: G negative (wild type),

    
    
        A homozygous (mutant)
        GA heterozygous
    
    









TABLE 41







Sequence, length and mass of extension


primer, extended primers and pause product


for HFE FM6-E assay, mutation C282Y (G845A)










Allele
Sequence
Length
Mass





FM6-E
GCCTGGGTGCTCCACCTGG
19 bp
5796.8 Da


 


G (Cyt)
GCCTGGGTGCTCCACCTGGC
20 bp
6070.0 Da


 


A (Tyr)
GCCTGGGTGCTCCACCTGGTA
21 bp
6398.2 Da


 


Pause
GCCTGGGTGCTCCACCTGGT
20 bp
6101.0 Da





(SEQ ID NOS: 893, 894, 895, and 896)






Assay: HFE-E6

Overview:















Wild type template




(SEQ ID NO: 897)


 


Mutated template




(SEQ ID NO: 898)


 


Extension primer




(SEQ ID NO: 51)









Amplification by PCR*:











HFE-F9
(SEQ ID NO: 49)









5′-ACGTTGGATGTAATGGGGATGGGACCTACC-3′



 










HFE-R10
(SEQ ID NO: 50)









5′-ACGTTGGATGTGCTCTCATCAGTCACATACC-3′






Amplicon length: 137 bp

    
    
        * amplification primers have the 10-mer tag specific for hME method:acgttggatg.
    
    


Extension:
Terminator Bases Mix: ACT










Extend Primer (HFE-E6):
(SEQ ID NO: 51)









5′ GGGAAGAGCAGAGATATACGT 3′



 










Analyte A:
(SEQ ID NO: 899)









5′ GGGAAGAGCAGAGATATACGTA 3′



 










Analyte G:
(SEQ ID NO: 900)









5′ GGGAAGAGCAGAGATATACGTGC 3′






Genotype Calls: G negative (wild type)

    
    
        A homozygous (mutant)
        AG heterozygous
    
    









TABLE 42







Sequence, length and mass of extension


primer, extended primers and pause product


for HFE HFE-E6 assay, mutation C282Y (G845A)










Allele
Sequence
Length
Mass





HFE-E6
GGGAAGAGCAGAGATATACGT
21 bp
6568.3 Da


 


G (Cyt)
GGGAAGAGCAGAGATATACGTGC
23 bp
7170.7 Da


 


A (Tyr)
GGGAAGAGCAGAGATATACGTA
22 bp
6865.5 Da


 


Pause
GGGAAGAGCAGAGATATACGTG
22 bp
       Da





(SEQ ID NOS: 901, 902, 903, and 904)






HFE S65C (A193T) Mutation in Exon 2:

This section describes the amplification and detection extension primers for the A193T Assay and provides the sequences of the primers. The sequences of the resulting extended primers are provided in Tables 35 and 36, below.
Assay: HFE S65C_E1
Overview:














Wild type template




(SEQ ID NO: 905)


 


Mutated template




(SEQ ID NO: 906)


 


Extension primer




(SEQ ID NO: 907)









Amplification by PCR*:











HFE-F1
(SEQ ID NO: 52)









5′-ACGTTGGATGATGACCAGCTGTTCGTGTTC-3′



 










HFE-R2
(SEQ ID NO: 53)









5′-ACGTTGGATGTCTACTGGAAACCCATGGAG-3′






Amplicon length: 74 bp

    
    
        * amplification primers have the 10-mer tag specific for hME method: acgttggatg.
    
    


Extension:
Terminator Bases Mix: CGT










Extend Primer (HFE S65C_E1):
(SEQ ID NO: 54)









5′ GGGCTCCACACGGCGAC 3′



(reverse primer)



 










Analyte A:
(SEQ ID NO: 908)









5′ GGGCTCCACACGGCGACT 3′



(reverse primer)



 










Analyte T:
(SEQ ID NO: 909)









5′ GGGCTCCACACGGCGACAC 3′



(reverse primer)






Genotype Calls: A negative (wild type)

    
    
        T homozygous (mutant)
        AT heterozygous
    
    









TABLE 43







Sequence, length and mass of


extension primer, extended primers and pause


product for HFE S65C-E1 assay, mutation A193T










Allele
Sequence
Length
Mass





HFE S65C_E1
GGGCTCCACACGGCGAC
17 bp
5181.4 Da


 


A NEGATIVE
GGGCTCCACACGGCGACT
18 bp
5469.6 Da


 


T HOMOZYGOUS
GGGCTCCACACGGCGACAC
19 bp
5767.8 Da


 


Pause
GGGCTCCACACGGCGACA
18 bp
       Da





(SEQ ID NOS: 910, 911, 912, and 913)






Assay: HFE S65C_E5

Overview:














Wild type template




(SEQ ID NO: 914)


 


Mutated template




(SEQ ID NO: 915)


 


Extension primer




(SEQ ID NO: 57)









Amplification by PCR*:










HFE-F7
(SEQ ID NO: 55)









5′-ACGTTGGATGTTCATGGGTGCCTCAGAGC-3′



 










HFE-R8
(SEQ ID NO: 56)









5′-ACGTTGGATGCCACATCTGGCTTGAAATTC-3′






Amplicon length: 153 bp

    
    
        * amplification primers have the 10-mer tag specific for hME method:acgttggatg (SEQ ID NO:916)
    
    


Extension:
Terminator Bases Mix: CGT








Extend Primer (HFE S65C_E5):
(SEQ ID NO: 57)


5′ TTCGTGTTCTATGATCATGAG 3′
(6442.2 Da)


 









Analyte A:

(SEQ ID NO: 917)








5′ TTCGTGTTCTATGATCATGAGAG 3′
(7068.6 Da)


 









Analyte T:

(SEQ ID NO: 918)








5′-TTCGTGTTCTATGATCATGAGT-3′
(6730.4 Da)






Genotype Calls: A negative (wild type)

    
    
        T homozygous (mutant)
        AT heterozygous
    
    









TABLE 44







Sequence, length and mass of extension


primer, extended primers and pause


product for HFE S65C-E5 assay, mutation A193T










Allele
Sequence
Length
Mass





HFE
TTCGTGTTCTATGATCATGAG
21 bp
6442.2 Da


S65C_E5





 


A
TTCGTGTTCTATGATCATGAGAG
23 bp
7068.6 Da


NEGATIVE





 


T HOMO-
TTCGTGTTCTATGATCATGAGT
22 bp
6730.4 Da


ZYGOUS





 


Pause
TTCGTGTTCTATGATCATGAGA
22 bp
       Da





(SEQ ID NOS: 919, 920, and 512-513, respectively)






Testing Procedure

Blood specimens and quality control procedures were performed according to protocols described in Example 1. Table 45, below, provides a list of controls used in the presently described assay.







TABLE 45





Controls Used in the Assays

















HFE H63D












NA13591
G
HFE H63D
homozygous mutant



393547
C
HFE H63D
negative- wild type



GM7798
C
HFE H63D
negative- wild type



GM8810
C
HFE H63D
negative- wild type



GM9729
C
HFE H63D
negative- wild type



NA14646
C
HFE H63D
negative- wild type



NA14702
C
HFE H63D
negative- wild type



NA16028
C
HFE H63D
negative- wild type



GM12783
CG
HFE H63D
heterozygous



NA14641
CG
HFE H63D
heterozygous



NA14650
CG
HFE H63D
heterozygous



NA16000
CG
HFE H63D
heterozygous









HFE C282Y












393547
A
HFE C282Y
homozygous mutant



NA14646
A
HFE C282Y
homozygous mutant



GM5896
G
HFE C282Y
negative- wild type



GM6023
G
HFE C282Y
negative- wild type



GM7798
G
HFE C282Y
negative- wild type



GM8810
G
HFE C282Y
negative- wild type



NA13591
G
HFE C282Y
negative- wild type



NA14702
G
HFE C282Y
negative- wild type



NA16000
G
HFE C282Y
negative- wild type



NA16028
G
HFE C282Y
negative- wild type



NA14641
AG
HFE C282Y
heterozygous



NA14650
AG
HFE C282Y
heterozygous









HFE S65C












393547
A
HFE S65C
negative- wild type



GM12783
A
HFE S65C
negative- wild type



GM5896
A
HFE S65C
negative- wild type



GM6023
A
HFE S65C
negative- wild type



GM7798
A
HFE S65C
negative- wild type



GM8810
A
HFE S65C
negative- wild type



GM9729
A
HFE S65C
negative- wild type



NA13591
A
HFE S65C
negative- wild type



NA14641
A
HFE S65C
negative- wild type



NA14646
A
HFE S65C
negative- wild type



NA14650
A
HFE S65C
negative- wild type



NA14702
A
HFE S65C
negative- wild type



NA16000
A
HFE S65C
negative- wild type



NA16028
AT
HFE S65C
heterozygous










PCR, dephosphorylation, homogenous MASSEXTEND (HME), and spectroclean resin clean up procedures were carried out as described in Example 1, above.
Results Analysis
Results were analyzed as described in Example 1, above. FIG. 7 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for H63D Histidine to Aspartic acid (C187G) mutation in the FM3-E assay. FIG. 8 is a mass spectrum of heterozygous “GC” alleles (heterozygous positive) for H63D Histidine to Aspartic acid (C187G) mutation in the HFE-E3 assay. FIG. 9 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for S65C Serine to Cysteine (A193T) mutation in the HFE S65C E1 assay. FIG. 10 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for S65C Serine to Cysteine (A193T) mutation in the FIFE S65C E5 assay. FIG. 11 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for C282Y cysteine to tyrosine (G845A) mutation in the FM6-E assay. FIG. 12 is a mass spectrum of heterozygous “GA” alleles (heterozygous positive) for C282Y cysteine to tyrosine (G845A) mutation in the HFE-E6 assay.
Exemplary test results are provided in Tables 46 to 48, below.
Validation Tests Results







TABLE 46







H63D mutation tested by HFE-E3_FM3-E assays











Source of



Low


DNA/Assay
Conservative
Moderate
Aggressive
Probability










ARUP 131 specimens











FM3-E
130
1
0
0


HFE-E3
123
7
1
0









Distribution of Genotypes:

















GG HOMOZYGOUS
N = 5



CC NEGATIVE
N = 68



GC HETEROZYGOUS
N = 58










Summary:

    
    
        a) FIFE assays, FM3-E and HFE-E3, were validated in two runs “DD33” and “Validation HFE-E3_FM3-E” for a total of 131 samples.
        b) The samples were obtained from ARUP and our results are compared to the results from ARUP.
        c) The results from 129 samples were in accordance with ARUP results for both assays.
        d) For two samples, 01hFE—021220 & 101hFE—021220, our results differ from those from ARUP. The sample: 101hFE—021220 is a duplicate (re-extracted and PCRed a second time) of the sample 01hFE—021220. All 4 of our results agreed. We suggest that our results are correct.
        e) Thirteen samples which did not give any result in one of the assays were re-run in subsequent runs and results were obtained for all the samples.
    
    









TABLE 47







C282Y mutation tested by FM6-E and HFE-E6 Assays











Source of



Low


DNA/Assay
Conservative
Moderate
Aggressive
Probability










ARUP 124 specimens











FM6-E
124
0
0
0


HFE-E6
122
2
0
0









Distribution of Genotypes:
















GG HOMOZYGOUS
N = 32



CC NEGATIVE
N = 47



GC HETEROZYGOUS
N = 45










Summary:

    
    
        a) HFE assays, FM6-E and HFE-E6, were validated in the run “VALIDATION HFE-FM6 HFE-E 041603” a total of 124 samples.
        b) The samples were obtained from ARUP and our results are compared to the results from ARUP.
        c) The results from 122 samples agreed with ARUP results for both assays.
        d) For two samples (04hFE—021220& 104hFE—021220) our results differ from those from ARUP. Sample 104hFE—021220 is a re-extracted second time sample 04hFE—021220, and all 4 of our results agree. Therefore, we suggest that our results are correct.
    
    









TABLE 48







S65C mutation tested by HFE S65C_E1


and HFE S65C_E5 assays











Source of



Low


DNA/Assay
Conservative
Moderate
Aggressive
Probability










ARUP 102 specimens











HFE S65C_E1
102
0
0
0


HFE S65C_E5
99
3
0
0









Distribution of Genotypes:

















TT HOMOZYGOUS
N = 0



AA NEGATIVE
N = 98



AT HETEROZYGOUS
N = 4










Summary:

    
    
        a) HFE assays, HFE S65C_E1 and HFE S65C_E5, were at total of 103 samples.
        b) The samples were obtained from ARUP and our results are compared to the results from ARUP.
        c) The results from 98 samples agree with ARUP results for both assays.
        d) For six samples (01hFE—021220 & 101hFE—021220, 04hFE—021220 & 104hFE—021220, 41hFE—021126 & 141hFE—021126) our results differ from those from ARUP. The samples: 101hFE—021220, 104hFE—021220, and 141hFE—021126 are duplicates (re-extracted and PCRed second time) of the samples 01hFE—021220, 04hFE—021220, and 41hFE—021126. All 4 of our results agree for each sample.
        e) In addition, since samples 01hFE—021220 and 04hFE—021220 had different calls for two mutations suggesting a mixup in the ARUP samples.
    
    


Other Embodiments
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

## Claims

1. A fully automated system for performing an assay on a biological sample, comprising:
(a) a sample transfer module for transferring a portion of the sample to a well of a first multi-well plate and programmed to exchange with at least one other module of the system information about the location of the portion in the first multi-well plate;
(b) a nucleic acid extraction module for extracting nucleic acids from cells within the portion and for transferring the portion from the first multi-well plate to a well of a second multi-well plate;
(c) a nucleic acid measurement module for measuring the concentration of nucleic acids in the portion;
(d) a PCR preparation module for adding polymerase chain reaction (PCR) reaction materials to the portion and programmed to receive information from (a) about the location of the portion in the first or second multi-well plate, and wherein the PCR preparation module comprises the primer set SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO. 15;
(e) a thermocyling module for amplifying a target sequence and extending a primer in the portion;
(f) a primer extension preparation module for adding primer extension reaction materials to the portion;
(g) a mass spectrometry preparation module for moving a sample of the portion to a support for analysis by mass spectrometry;
(h) a mass spectrometry module for analyzing the sample;
(i) a central controller:
(1) in communication with, and programmed to exchange information about the biological sample with, an outside system or database; and
(2) in communication with, and programmed to exchange information about the biological sample with, at least (a)-(h) of the system; and
(j) plate editor software programmed to a multi-well plate where the portion will be located prior to dispensing of the portion into a well of the multi-well plate, exchange information with at least (a), (d) and (i) of the system and an outside system or database, exchange information with (a) and (d) about which well in a multi-well plate will receive or contains the portion, and exchange information with (d) about which assay to perform on the portion.