➤ Get the DrugPatentWatch Daily Briefing

Get Daily Updates on Generic Entry, Litigation, Biosimilars, and more …

Serving leading biopharmaceutical companies globally:

Medtronic
Dow
Express Scripts
Merck
Baxter
Mallinckrodt

Last Updated: September 18, 2021

DrugPatentWatch Database Preview

Claims for Patent: 5,545,527

➤ Get the DrugPatentWatch Daily Briefing

« Back to Dashboard

Summary for Patent: 5,545,527
Title: Method for testing for mutations in DNA from a patient sample
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.
Inventor(s): Stevens; John K. (Mississauga, CA), Dunn; James M. (Scarborough, CA)
Assignee: Visible Genetics Inc. (Toronto, CA)
Application Number:08/271,946
Patent Claims:1. 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 each 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 the 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 either 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.

2. A method according to claim 1, wherein the antibody binds to the protein gene product of the wild-type gene of interest, and the observation of immunological product at levels below a standard level which results from tests on homozygous wild-type samples is indicative of the presence of a mutation.

3. A method according to claim 1, wherein the antibody binds to a mutant gene product caused by the disease-associated mutation and not to the wild-type gene product of the gene of interest, and the observation of an immunological product is indicative of the presence of the disease associated mutation.

4. A method according to claim 1, wherein the sequences of a plurality of regions in the gene of interest are determined.

5. A method according to claim 4, wherein the sequences of the plurality of regions are determined in at least two phases in which regions having a greater incidence of disease-associated mutations in a population group to which the patient belongs are tested before regions having a lower incidence of mutations.

6. 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 each 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 indicated the absence 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 either 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.

7. 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 portion of each patient sample with a nucleic acid probe which specifically and selectively hybridizes with the gene of interest in either 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 of the mutation, respectively, 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 wild-type or mutant forms of the gene of interest.

8. 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.

9. A method according to claim 8, wherein the first assay is an immunoassay.

10. A method according to claim 8, wherein the nucleic acid sequence of at least one region of the gene of interest is determined in course of performing the final assay.

11. A method according to claim 10, wherein the first assay is an immunoassay.

12. A method according to claim 8, 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 immediately 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.

13. A method according to claim 12, wherein the length of the amplification products is determined by electrophoresis on a sequencing gel.

14. A method according to claim 13, wherein the sequencing gel has a resolution of one base pair.

15. A method according to claim 14, wherein the sequencing gel is a polyacrylamide gel.

16. A method according to claim 12, wherein the primers are each coupled to a detectable label.

17. A method according to claim 16, wherein the detectable label is fluorescein.

18. A method according to claim 12, wherein a plurality of exons of the gene of interest are coamplified in a single amplification reaction.

19. A method according to claim 18, wherein the primer pairs for each coamplified exon are selected to have a common melting temperature and to produce amplification products having differing lengths.

20. A method according to claim 8, wherein the cost per sample for performing the first assay is less than the cost per sample for performing the final assay.

21. A method according to claim 8, wherein the hierarchy further includes an intermediate assay for detecting the presence of the disease-associated mutation which is performed after the first assay on samples for which the first assay did not indicate the presence of a mutation and before the final assay, and wherein the final assay is performed only if the intermediate assay did not indicate the presence of a mutation, said intermediate assay providing essentially no false positives, and fewer false negatives than the first assay.

22. A method according to claim 21, wherein the first assay is an immunoassay.

23. A method according to claim 22, wherein the intermediate assay is a nucleic acid hybridization probe-based assay.

24. A method according to claim 23, wherein the sequence of a region of a gene of interest is sequenced in course of performing the final assay.

25. A method for generating a report on the presence or absence of a disease-associated mutation in a patient sample, comprising the steps of:

(a) selecting a hierarchy of molecular assay techniques comprising at least a first molecular assay and 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 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 assay does not indicate the presence of a disease-associated mutation,

(c) analyzing the patient sample using the final molecular assay; and

(d) generating a report containing advice that the disease-associated mutation is present in the patient sample if a test result indicating the presence of the disease-associated mutation is obtained in any assay of the hierarchy, and advice that the disease-associated mutation is absent if a test result indicating the absence of a mutation is obtained in all of the assays in the hierarchy.

26. A method according to claim 25, wherein the report is a printed report.

27. A method according to claim 25, wherein the report is an electronic communication.

28. A method according to claim 25, wherein the report is a data entry in a computer record containing information about the patient.

29. A method according to claim 25, wherein the first assay is an immunoassay.

30. A method according to claim 25, wherein the nucleic acid sequence of at least one region of the gene of interest is determined in course of performing the final assay.

31. A method according to claim 28, wherein the first assay is an immunoassay.

32. A method according to claim 25, 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.

33. A method according to claim 30, wherein the length of the amplification products is determined by electrophoresis on a sequencing gel.

34. A method according to claim 31, wherein the sequencing gel has a resolution of one base pair.

35. A method according to claim 32, wherein the sequencing gel is a polyacrylamide gel.

36. A method according to claim 30, wherein the primers are each coupled to a detectable label.

37. A method according to claim 34, wherein the detectable label is fluorescein.

38. A method according to claim 30, wherein a plurality of exons of the gene of interest are coamplified in a single amplification reaction.

39. A method according to claim 36, wherein the primer pairs for each coamplified exon are selected to have a common melting temperature and to produce amplification products having differing lengths.

40. A method according to claim 25, wherein the cost per sample for performing the first assay is less than the cost per sample for performing the final assay.

41. A method according to claim 25, wherein the hierarchy further includes an intermediate assay for detecting the presence of the disease-associated mutation which is performed after the first assay on samples for which the first assay did not indicate the presence of a mutation and before the final assay, and wherein the final assay is performed only if the intermediate assay did not indicate the presence of a mutation, said intermediate assay providing essentially no false indications of the presence of a mutation, and fewer false indications of the absence of a mutation than the first assay.

42. A method according to claim 41, wherein the first assay is an immunoassay.

43. A method according to claim 42, wherein the intermediate assay is a nucleic acid hybridization probe-based assay.

44. A method according to claim 41, wherein the sequence of a region of a gene of interest is sequenced in course of performing the final assay.

Details for Patent 5,545,527

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04 ⤷  Free Forever Trial Visible Genetics Inc. (Toronto, CA) 2039-02-26 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04 ⤷  Free Forever Trial Visible Genetics Inc. (Toronto, CA) 2039-02-26 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04 ⤷  Free Forever Trial Visible Genetics Inc. (Toronto, CA) 2039-02-26 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

Make Better Decisions: Try a trial or see plans & pricing

Serving leading biopharmaceutical companies globally:

Colorcon
Moodys
AstraZeneca
McKesson
Harvard Business School
Boehringer Ingelheim

Drugs may be covered by multiple patents or regulatory protections. All trademarks and applicant names are the property of their respective owners or licensors. Although great care is taken in the proper and correct provision of this service, thinkBiotech LLC does not accept any responsibility for possible consequences of errors or omissions in the provided data. The data presented herein is for information purposes only. There is no warranty that the data contained herein is error free. thinkBiotech performs no independent verification of facts as provided by public sources nor are attempts made to provide legal or investing advice. Any reliance on data provided herein is done solely at the discretion of the user. Users of this service are advised to seek professional advice and independent confirmation before considering acting on any of the provided information. thinkBiotech LLC reserves the right to amend, extend or withdraw any part or all of the offered service without notice.