CA2369045A1 - Methods for improving sensitivity and specificity of screening assays - Google Patents

Methods for improving sensitivity and specificity of screening assays Download PDF

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Publication number
CA2369045A1
CA2369045A1 CA002369045A CA2369045A CA2369045A1 CA 2369045 A1 CA2369045 A1 CA 2369045A1 CA 002369045 A CA002369045 A CA 002369045A CA 2369045 A CA2369045 A CA 2369045A CA 2369045 A1 CA2369045 A1 CA 2369045A1
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Canada
Prior art keywords
sample
primer
nucleic acid
assay
bodily
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Granted
Application number
CA002369045A
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French (fr)
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CA2369045C (en
Inventor
Stanley N. Lapidus
Anthony P. Shuber
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Esoterix Genetic Laboratories LLC
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Individual
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Publication of CA2369045C publication Critical patent/CA2369045C/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Abstract

Methods of the invention comprise assays for markers indicative of cancer or precancer. Assays of the invention are performed on samples obtained from a patient by non-invasive or minimally-invasive methods. The invention provides nucleic acid indicia of cancer or precancer with high sensitivities and high specificities for detection.

Claims (33)

1. A method of screening a human patient for cancer or precancer, the method comprising the steps of:
(a) determining, in a patient sample of a bodily excretion or a bodily fluid, the amount of DNA greater than about 200 by in length; and (b) comparing said amount to the amount of DNA greater than about 200 by in length expected to be present in a sample obtained from a healthy patient, wherein a statistically significant larger amount of nucleic acids greater than about 200 by in length in said patient sample indicates a positive screen.
2. The method of claim 1, further comprising the step of amplifying DNA in said sample of bodily excretion or bodily fluid using primers spaced about 200 by apart.
3. The method of claim 1, further comprising the step of determining a ratio of said first amount of DNA to an amount of DNA having a length less than about 200 by in said sample of bodily excretion or bodily fluid.
4. The method of claim 1, further comprising the step of:
(a) selecting one or more informative nucleic acid mutations; and (b) performing an assay to detect the presence or absence of at least one of said mutations in said sample.
5. The method of claim 1, further comprising the step of determining an amount of amplifiable nucleic acid in said sample of bodily excretion or bodily fluid.
6. The method of claim 1, further comprising the step of detecting at least one nucleic acid mutation in said sample of bodily excretion or bodily fluid.
7. The method of claim 1, further comprising the step of performing a plurality of nucleic acid assays.
8. The method of claim 7, wherein said plurality comprises 2 to 5 assays.
9. The method of claim 7, wherein each assay detects a different nucleic acid marker.
10. The method of claim 7, wherein said assays are selected from the group consisting of multiple mutation detection, quantitative polymerase chain reaction, sequence-specific hybrid capture, oligo-ligation, amplification refractory mutation system, single-stranded conformational polymorphism detection, sequencing, mismatch detection, and single base extension.
11. The method of claim 1, further comprising the step of performing gel electrophoresis on nucleic acids obtained from said sample of bodily excretion or bodily fluid.
12. The method of claim 6, wherein said step of detecting at least one nucleic acid mutation further comprises performing a single base extension assay.
13. The method of any of claims 1, 2, or 6, wherein said method of screening provides a sensitivity of detection of at least 60% and a specificity of detection of at least 90%.
14. The method of claim 13, wherein said sensitivity and said specificity are determined by comparing results obtained in said screening method with results obtained using an invasive diagnostic method.
15. The method of claim 7, wherein at least one of said assays is an assay to detect loss of heterozygosity in at least a portion of a chromosomal arm.
16. The method of any of claims 1, 2, or 6, wherein said precancer is an adenoma.
17. The method of any of claims 1, 2, or 6, wherein said bodily excretion is selected from the group consisting of stool, and a homogenate of stool.
18. The method of any of claims 1, 2, or 6, wherein said bodily excretion or bodily fluid is selected from the group consisting of pus, sputum, urine, blood, cerebrospinal fluid, semen, and aspirate.
19. The method of claim 12, wherein said single base extension assay comprises the steps of:
(a) annealing an oligonucleotide primer to a nucleic acid sample under conditions that promote exact complementary hybridization between said primer and a portion of a nucleic acid in said sample;
(b) extending said primer by a single base;
(c) separating said extended primer from said portion; and (d) identifying the base incorporated into said extended primer, hereby to identify said single base.
20. The method of claim 12, wherein said single base extension assay comprises the steps of:
(a) annealing an oligonucleotide primer to a nucleic acid sample under conditions that promote exact complementary hybridization between said primer and a portion of a nucleic acid in said sample;
(b) exposing said sample to two different deoxynucleotides under conditions to promote primer extension;
(c) extending said primer until said primer can no longer be extended;
(d) separating said extended primer from said portion; and (e) identifying the base incorporated into said extended primer, thereby to identify said single base.
21. The method of claim 20, wherein at least one of said deoxynucleotides is detectably labeled.
22. The method of claim 12, wherein said single base extension assay comprises identifying single nucleotides in each of a plurality of genomic loci.
23. The method of claim 22, wherein said genomic loci are selected from the group consisting of apc, Kras, p53, and bat-26.
24. The method of claim 7, wherein said plurality of assays comprises a quantitative polymerase chain reaction assay and an assay for a mutation in bat-26.
25. The method of claim 24, wherein said assay for a mutation in bat-26 comprises a primer extension assay to identify fragments of said bat-26.
26. The method of any of claims 1, 2, or 6, wherein said precancer is an adenoma.
27. The method of any of claims 1, 2, or 6, wherein said cancer is colorectal cancer.
28. The method of claim 13, wherein said sensitivity and said specificity are determined by comparing results obtained in said screening method with results obtained using an invasive diagnostic method.
29. The method of claim 28, wherein said invasive diagnostic method is a colonoscopy.
30. The method of claim 28, wherein said invasive diagnostic method is conducted contemporaneously with said screening method.
31. The method of claim 28, wherein said invasive diagnostic method is conducted within from about 3 hours to about 3 months of the time at which said screening method is conducted.
32. The method of claim 15, wherein said assay to detect loss of heterozygosity is selected from the group consisting of enumerative LOH and single base extension.
33. The method of claim 15, wherein said chromosomal arm is selected from the group consisting of chromosome 17p, chromosome 5p, chromosome 8p, chromosome 1q, and chromosome 18q.
CA2369045A 1999-03-26 2000-03-24 Methods for improving sensitivity and specificity of screening assays Expired - Fee Related CA2369045C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09/277,016 US6143529A (en) 1996-08-14 1999-03-26 Methods for improving sensitivity and specificity of screening assays
US09/277,016 1999-03-26
PCT/US2000/007882 WO2000058514A2 (en) 1999-03-26 2000-03-24 Methods for improving sensitivity and specificity of screening assays

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CA2369045A1 true CA2369045A1 (en) 2000-10-05
CA2369045C CA2369045C (en) 2010-06-01

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CA2369045A Expired - Fee Related CA2369045C (en) 1999-03-26 2000-03-24 Methods for improving sensitivity and specificity of screening assays

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US (1) US6143529A (en)
EP (1) EP1185693B1 (en)
JP (3) JP2002539848A (en)
AT (1) ATE337413T1 (en)
AU (1) AU761722B2 (en)
CA (1) CA2369045C (en)
DE (1) DE60030281T2 (en)
WO (1) WO2000058514A2 (en)

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Also Published As

Publication number Publication date
DE60030281D1 (en) 2006-10-05
ATE337413T1 (en) 2006-09-15
EP1185693A2 (en) 2002-03-13
JP2010172347A (en) 2010-08-12
EP1185693B1 (en) 2006-08-23
CA2369045C (en) 2010-06-01
US6143529A (en) 2000-11-07
JP2002539848A (en) 2002-11-26
WO2000058514A2 (en) 2000-10-05
JP2013116131A (en) 2013-06-13
DE60030281T2 (en) 2007-08-30
AU761722B2 (en) 2003-06-05
WO2000058514A3 (en) 2001-12-13
AU3918900A (en) 2000-10-16

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