US 6,270,963 B1Granted

Method for testing for mutations in DNA from a patient sample

AssigneeVisible Genetics Inc.

InventorsJohn K. Stevens, James M. Dunn, Denis Capatos, David E. Matthews

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Abstract

A hierarchy of at least two assay techniques is utilized in testing for disease-associated mutations. The first assay in the hierarchy is selected to provide a highly specific test for the existence of the disease-associated mutation, although the accuracy of the test need not be high. The final assay in the hierarchy is selected to provide a highly accurate and highly specific test for the existence of the disease associated mutation. Intermediate tests of progressively greater accuracy may also be included in the hierarchy. Once the hierarchy has been selected for a given mutation-associated disease, a patient sample is analyzed the patient sample using the first, lowest accuracy assay in the hierarchy. If the result of the first assay is negative for the presence of a disease-associated mutation, then the next assay in the hierarchy is performed. This process is repeated until the final assay has been performed on all samples which gave negative results when tested by all less-accurate assays in the hierarchy. The test may be used for diagnosis and targeted screening for p53 mutations and mutations in the RB1 gene.

Claims23

Dependent claims

A method for testing a plurality of patients for a disease-associated mutation in a gene of interest comprising the steps of:

(a) performing an immunoassay on samples obtained from each of the plurality of patients by combining a portion of sample with an antibody which forms an immunological reaction product by binding to a protein gene product of the gene of interest and monitoring for formation of the immunological reaction product, said antibody being selected to provide fewer than 1% false results indicating the presence of a mutation when combined with the protein gene product of the gene of interest, whereby the formation of an immunological product is indicative of the presence or absence of a mutation in the gene of interest;
(b) performing a probe-based assay on each patient sample for which the results of the immunoassay did not indicate the presence of a mutation, but not on those samples for which the immunoassay indicated the presence of the mutation by combining a second portion of each patient sample with a nucleic acid probe which specifically and selectively hybridizes with the gene of interest in its mutated or wild-type form, whereby the formation of a duplex nucleic acid hybrid containing the nucleic acid probe is indicative of the presence or absence, respectively, of the mutation in the gene of interest;
and
(c) determining the sequence of DNA in at least one selected region of the gene of interest on each patient sample for which the results of the probe-based assay did not indicate the presence of the mutation and comparing the sequence determined with known sequences of normal or mutant forms of the gene of interest,
wherein the immunoassay and probe-based assay used are selected by a method comprising the steps of:
identifying a plurality of immunoassays and probe-based assays for the disease-associated mutation;
determining the level of false results for each assay by comparing results from assays on a statistically significant plurality of samples against results from assays on the same samples using a test protocol accepted as having essentially no false results;
defining an outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of assays to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;
determining overall sensitivity overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;
determining the estimated cost of testing for those combinations where the overall sensitivity overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet a predefined threshold;
and
selecting a diagnostic algorithm of assays based upon the low estimated cost of testing.

9 more elements

A method for testing a plurality of patients for a disease-associated mutation in a gene of interest comprising the steps of:

(a) performing an immunoassay on samples obtained from each of the plurality of patients by combining a portion of sample with an antibody which forms an immunological reaction product by binding to a protein gene product of the gene of interest and monitoring for formation of the immunological reaction product, said antibody being selected to provide fewer than 1% false results indicating the presence of a mutation when combined with the protein gene product of the gene of interest, whereby the formation of an immunological product is indicative of the presence or absence of the mutation in the gene of interest;
and
(b) performing a probe-based assay on each patient sample for which the results of the immunoassay did not indicate the presence of a mutation, but not for those samples for which the immunoassay indicated the presence of the mutation by combining a second portion of each patient sample with a nucleic acid probe which specifically and selectively hybridizes with the gene of interest in its mutated or wild-type form, whereby the formation of a duplex nucleic acid hybrid containing the nucleic acid probe is indicative of the presence or absence, respectively, of the mutation in the gene of interest,
wherein the immunoassay and probe-based assay used are selected by a method comprising the steps of:
identifying a plurality of immunoassays and probe-based assays for the disease-associated mutation;
determining the level of false results for each assay by comparing results from assays on a statistically significant plurality of samples against results from tests on the same samples using a test protocol accepted as having essentially no false results;
defining an outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of tests to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;
determining overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;
determining the estimated cost of testing for those combinations where the overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet the predefined threshold;
and
selecting a diagnostic algorithm of tests based upon the low estimated cost of testing.

8 more elements

A method for testing a plurality of patients for a disease-associated mutation in a gene of interest comprising the steps of:

(a) performing a probe-based assay on each patient sample by combining a second portion of each patient sample with a nucleic acid probe which specifically and selectively hybridizes with the gene of interest in its mutant or wild-type form said probe-based assay providing essentially no false indications of the presence of a mutation, whereby the formation of a duplex nucleic acid hybrid containing the nucleic acid probe is indicative of the presence or absence, respectively, of the mutation in the gene of interest;
and
(b) determining the sequence of DNA in at least a selected region of the gene of interest on each patient sample for which the results of the probe-based assay did not indicate the presence of a mutation, but not on those samples for which the probe-based assay indicated the presence of the mutation and comparing the sequence determined with known sequences of normal or mutant forms of the gene of interest,
wherein the probe-based assay used is selected by a method comprising the steps of:
identifying a plurality of probe-based assays for the disease-associated mutation;
determining the level of false results for each assay by comparing results from assays on a statistically significant plurality of samples against results from assays on the same samples using a test protocol accepted as having essentially no false results;
defining an outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of tests to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;
determining overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;
determining the estimated cost of testing for those combinations where the overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet the predefined threshold;
and
selecting a diagnostic algorithm of assays based upon the low estimated cost of testing.

8 more elements

A method for testing a patient sample for the presence or absence of a disease-associated mutation in a gene of interest in a patient sample, comprising the steps of:

(a) selecting a hierarchy of molecular assay techniques comprising at least a first molecular assay and a final molecular assay, said first molecular assay being selected to provide a test for the existence of the disease-associated mutation with essentially no false results indicating the presence of a mutation and said final molecular assay being selected to provide a test for the existence of the disease associated mutation with essentially no false results indicating the presence or absence of a mutation;
(b) analyzing the patient sample using the first assay;
and
(c) if the result of the first assay did not unambiguously indicate the presence of a disease-associated mutation, analyzing the patient sample using the final assay, wherein at least one assay in the hierarchy comprises the steps of
quantitatively amplifying one or more exons of the gene of interest in the sample using primers complementary to intron regions flanking each amplified exon;
and
determining the lengths of the amplification products for each amplified sample exon and comparing that length to the length of amplification products obtained when a wild-type gene is amplified using the same primers, whereby differences in length between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence of an insertion or deletion mutation in the gene of interest in the sample,
and wherein the assays used are selected by a method comprising the steps of:
identifying a plurality of assays for the disease-associated mutation;
determining the level of false results for each assay by comparing results from assays on a statistically significant plurality of samples against results from assays on the same samples using a test protocol accepted as having essentially no false results;
defining an outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of tests to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;
determining overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;
determining the estimated cost of testing for those combinations where the overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet the predefined threshold;
and
selecting a diagnostic algorithm of assays based upon the low estimated cost of testing.

12 more elements

A method for testing a patient sample for a disease-associated mutation in a gene of interest, comprising the steps of:

(a) selecting a hierarchy of molecular assay techniques comprising at least a first molecular assay and a final molecular assay, said first molecular assay being selected to provide a test for the existence of the disease-associated mutation with essentially no false results indicating the presence of a mutation, and said final molecular assay being selected to provide a test for the existence of the disease associated mutation with essentially no false results indicating the presence or absence of a mutation;
(b) analyzing the patient sample using the first molecular assay;
and, if the result of the first molecular assay did not unambiguously indicate the presence of the disease-associated mutation,
(c) analyzing the patient sample using the final molecular assay,
wherein the hierarchy of tests is selected by a method comprising the steps of:
identifying a plurality of tests for the disease-associated mutation;
determining the level of false results for each test by comparing results from tests on a statistically significant plurality of samples against results from tests on the same samples using a test protocol accepted as having essentially no false results;
defining a outcome mapping matrix containing a plurality of diagnostic algorithms each comprising a combination of tests to be evaluated and assessing the significance of all possible combinations of negative and positive results for the algorithms in the matrix;
determining overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result for at least some of the algorithms in the outcome matrix;
determining the estimated cost of testing for those combinations where the overall sensitivity, overall specificity, overall predictive value of a positive algorithm result and overall predictive value of a negative algorithm result meet the predefined threshold;
and
selecting a diagnostic algorithm of tests based upon the low estimated cost of testing.

9 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/750,232Filed 29 Nov 1996 · continuation in part
Priority: 8 Jul 1994
Granted: 7 Aug 2001
Term ends: Not estimatedThe source records don’t support an estimate.

Classification6 codes

C12Q 1/6827
Hybridisation assays: for detection of mutation or polymorphism
C12Q 1/6858
Nucleic acid amplification reactions: allele-specific amplification
C12Q 1/6886
For diseases caused by alterations of genetic material: for cancer
C12Q 2600/156
Oligonucleotides characterized by their use: polymorphic or mutational markers