Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-07-11
2002-08-27
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S007100, C435S091100, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06440706
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention is related to diagnostic genetic analyses. In particular it relates to detection of genetic changes and gene expression.
BACKGROUND OF THE INVENTION
In classical genetics, only mutations of the germ-line were considered important for understanding disease. With the realization that somatic mutations are the primary cause of cancer (
1
), and may also play a role in aging (
2
,
3
), new genetic principles have arisen. These discoveries have provided a wealth of new opportunities for patient management as well as for basic research into the pathogenesis of neoplasia. However, many of these opportunities hinge upon detection of a small number of mutant-containing cells among a large excess of normal cells. Examples include the detection of neoplastic cells in urine (
4
), stool (
5
,
6
), and sputum (
7
,
8
) of patients with cancers of the bladder, colorectum, and lung, respectively. Such detection has been shown in some cases to be possible at a stage when the primary tumors are still curable and the patients asymptomatic. Mutant sequences from the DNA of neoplastic cells have also been found in the blood of cancer patients (
9
-
11
). The detection of residual disease in lymph nodes or surgical margins may be useful in predicting which patients might benefit most from further therapy (
12
-
14
). From a basic research standpoint, analysis of the early effects of carcinogens is often dependent on the ability to detect small populations of mutant cells (
15
-
17
).
Because of the importance of this issue in so many settings, many useful techniques have been developed for the detection of mutations. DNA sequencing is the gold standard for the detection of germ line mutations, but is useful only when the fraction of mutated alleles is greater than ~20% (
18
,
19
). Mutant-specific oligonucleotides can sometimes be used to detect mutations present in a minor proportion of the cells analyzed, but the signal to noise ratio distinguishing mutant and wild-type (WT) templates is variable (
20
-
22
). The use of mutant-specific primers or the digestion of polymerase chain reaction (PCR) products with specific restriction endonucleases are extremely sensitive methods for detecting such mutations, but it is difficult to quantitate the fraction of mutant molecules in the starting population with these techniques (
23
-
28
). Other innovative approaches for the detection of somatic mutations have been reviewed (
29
-
32
). A general problem with these methods is that it is difficult or impossible to independently confirm the existence of any mutations that are identified.
Thus there is a need in the art for methods for accurately and quantitatively detecting genetic sequences in mixed populations of sequences.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods for determining the presence of a selected genetic sequence in a population of genetic sequences.
It is another object of the present invention to provide molecular beacon probes useful in the method of the invention.
These and other objects of the invention are achieved by providing a method for determining the presence of a selected genetic sequence in a population of genetic sequences. A biological sample comprising nucleic acid template molecules is diluted to form a set of assay samples. The template molecules within the assay samples are amplified to form a population of amplified molecules in the assay samples of the set. The amplified molecules in the assay samples of the set are then analyzed to determine a first number of assay samples which contain the selected genetic sequence and a second number of assay samples which contain a reference genetic sequence. The first number is then compared to the second number to ascertain a ratio which reflects the composition of the biological sample.
Another embodiment of the invention is a method for determining the ratio of a selected genetic sequence in a population of genetic sequences. Template molecules within a set comprising a plurality of assay samples are amplified to form a population of amplified molecules in each of the assay samples of the set. The amplified molecules in the assay samples of the set are analyzed to determine a first number of assay samples which contain the selected genetic sequence and a second number of assay samples which contain a reference genetic sequence. The first number is compared to the second number to ascertain a ratio which reflects the composition of the biological sample.
According to another embodiment of the invention, a molecular beacon probe is provided. It comprises an oligonucleotide with a stem-loop structure having a photoluminescent dye at one of the 5′ or 3′ ends and a quenching agent at the opposite 5′ or 3′ end. The loop consists of 16 base pairs and has a T
m
of 50-51° C. The stem consists of 4 base pairs having a sequence 5′-CACG-3′.
A second type of molecular beacon probe is provided in another embodiment. It comprises an oligonucleotide with a stem-loop structure having a photoluminescent dye at one of the 5′ or 3′ ends and a quenching agent at the opposite 5′ or 3′ end. The loop consists of 19-20 base pairs and has a T
m
of 54-56° C. The stem consists of 4 base pairs having a sequence 5′-CACG-3′.
Another embodiment provides the two types of molecular beacon probes, either mixed together or provided in a divided container as a kit.
The invention thus provides the art with the means to obtain quantitative assessments of particular DNA or RNA sequences in mixed populations of sequences using digital (binary) signals.
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patent: 5925517 (1999-07-01), Tyagi et al.
patent: 5928870 (1999-07-01), Lapidus et al.
patent: 6020137 (2000-02-01), Lapidus et al.
patent: 6037130 (2000-03-01), Tyagi et al.
patent: 6143496 (2000-11-01), Brown et al.
patent: 0643140 (1995-03-01), None
patent: WO 95/13399 (1995-05-01), None
patent: WO 99/13113 (1999-03-01), None
Newton Essential PCR pp. 51-52 1995.*
Darren G. Monckton, et al., “Minisatellite “Isoallele” Discrimination in Pseudohomozygotes by Single Molecule PCR and Variant Repeat Mapping”, Genomics 11, pp. 465-467, 1991.
Gualberto Ruano, et al., “Haplotype of Multiple Polymorphisms Resolved by Enzymatic Amplification of Single DNA Molecules”, Proc. National Science USA, 1990 vol. 87 pp 6296-6300.
W. Navidi, et al., “Using PCR in Preimplantation Genetic Disease Diagnosis”, Human Reproduction, vol. 6, No. 6, pp. 836-849, 1991.
Hongua Li, et al., “Amplification and Analysis of DNA Sequences in Single Human Sperm and Diploid Cells”, Nature, vol. 335, Sep. 29, 1988 pp. 414-417.
Ramon Parsons, et al., “Mismatch Repair Deficiency in Phenotypically Normal Human Cells”, Science, vol. 268, May 5, 1995 pp. 738-740.
Lin Zhang, et al., “Whole Genome Amplification from a Single Cell: Implications for Genetic Analysis”, Proc. National Science USA, vol. 89, pp. 5847-5851, Jul. 1992.
David Sidransky, et al., “Clonal Expansion of p53 Mutant Cells in Associated with Brain Tumor Progression”, Nature, Feb. 27, 1992 vol. 355, pp. 846-847.
Alec J. Jeffreys, et al., “Mutation Processes at Human Minisatellites”, Electophoresis, pp. 1577-1585, 1995.
C. Schmitt, et al., “High Sensitive DNA Typing Approaches for the Analysis of Forensic Evidence: Comparison of Nested Variable Number of Tandem Repeats (VNTR) Amplification and a Short Tandem Repeats (STR) Polymorphism”, Forensic Science International, vol. 66, pp. 129-141, 1994.
Paul M. Lizardi, et al., “Mutation Detection and Single-Molecule Counting Using Isothermal Rolling-Circle Amplification”, Nature Genetics, vol. 19, Jul. 1988 pp. 225-232.
A. Piatek et al., “Molecular Beacon Sequence Analysis for Detecting Drug Resistance in Mycobacterium Tuberculosis”, Nature Biotechnology, Apr. 1998, pp. 359-363, vol. 16, No. 4.
S. Tyagi et al., “Multicolor Molecular Beacons for allel
Kinzler Kenneth W.
Vogelstein Bert
Banner & Witcoff , Ltd.
Johns Hopkins University
Siew Jeffrey
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