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Systems and methods for analyzing nucleic acid sequences

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Systems and methods for analyzing nucleic acid sequences


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

Browse recent University Of Massachusetts patents - Shrewsbury, MA, US
Inventors: Edward I. Ginns, Marzena Galdzicka
USPTO Applicaton #: #20120270206 - Class: 435 5 (USPTO) - 10/25/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Virus Or Bacteriophage

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The Patent Description & Claims data below is from USPTO Patent Application 20120270206, Systems and methods for analyzing nucleic acid sequences.

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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 (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 IIa, 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 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 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.

FIG. 13A1-13A3 is a set of mass spectra in a CMV quantitative assay on samples containing 400 CMV copies/ml.

FIG. 13B1-13B3 is a set of mass spectra in a CMV quantitative assay on samples containing 4000 CMV copies/ml.

FIG. 13C1-13C3 is a set of mass spectra in a CMV quantitative assay on samples containing 40,000 CMV copies/ml.

FIG. 13D-13D3 is a set of mass spectra in a CMV quantitative assay on samples containing 400,000 CMV copies/ml.

FIG. 13E1-13E3 is a set of mass spectra in a CMV quantitative assay on samples containing 4,000,000 CMV copies/ml.

FIG. 13F1-13F3 is a set of mass spectra in a CMV quantitative assay on samples containing 40,000,000 CMV copies/ml.

FIG. 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.

FIG. 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



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stats Patent Info
Application #
US 20120270206 A1
Publish Date
10/25/2012
Document #
13337740
File Date
12/27/2011
USPTO Class
435/5
Other USPTO Classes
4352872, 435/612, 435/615
International Class
/
Drawings
51



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