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US 6,753,147 B2Granted
Digital amplification
AssigneeThe Johns Hopkins University
InventorsBert Vogelstein, Kenneth W. Kinzler
Life of this patent
Estimating the term…
Abstract
The identification of pre-defined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. The exponential, analog nature of the polymerase chain reaction is transformed into a linear, digital signal suitable for this purpose. Single molecules can be isolated by dilution and individually amplified; each product is then separately analyzed for the presence of pre-defined mutations. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.
Claims43
A method for detecting a target nucleic acid, the method comprising the steps of:
- separating a sample in which a target nucleic acid is present in an amount less than about 20% relative to non-target nucleic acid in said sample, to form a plurality of assay samples;
- amplifying said target nucleic acid in said assay samples;
- hybridizing the amplification product to a first molecular beacon probe which hybridizes to the target nucleic acid and to a second molecular beacon probe which hybridizes to the non-target nucleic acid, wherein each of the first and the second molecular beacon probes comprises a photoluminescent dye and a quenching agent at opposite 5′ and 3′ ends, wherein the photoluminescent dye on the first and second molecular beacon probes are different, and wherein the first molecular beacon probe further comprises a stem and a loop, wherein the stem comprises about 4 base pairs having a sequence 5′-CACG-3′, and the loop comprises about 16 base pairs and has a Tm of about 50-51° C.;
- and
- detecting the first and the second molecular beacon probes hybridized to the target nucleic acid, thereby detecting the target nucleic acid.
2 more elements
A method for detecting a target nucleic acid, the method comprising the steps of:
- providing a sample comprising X% of a target nucleic acid, wherein X is less than 100;
- dividing the sample to produce a plurality of assay samples;
- wherein the ratio of target to non-target nucleic acid in at least one of the samples is greater than X%;
- amplifying the target nucleic acid to form an amplification product;
- hybridizing the amplification product to a first molecular beacon probe which hybridizes to the target nucleic acid and a second molecular beacon probe which hybridizes to the non-target nucleic acid, wherein each of the first and the second molecular beacon probes comprises a photoluminescent dye and a quenching agent at opposite 5′ and 3′ ends, wherein the photoluminescent dye on the first and second molecular beacon probes are different, and wherein the first molecular beacon probe further comprises a stem and a loop, wherein the stem comprises about 4 base pairs having a sequence 5′-CACG-3′, and the loop comprises about 16 base pairs and has a Tm of about 50-51° C.;
- and
- detecting the first and second molecular beacon probes hybridized to the target nucleic acid, thereby detecting the target nucleic acid.
4 more elements
A method for detecting a target nucleic acid in a population of non-target nucleic acid contained in a sample, the method comprising:
- dividing a heterogenous sample comprising target nucleic acid and non-target nucleic acid to form a plurality of assay samples, wherein the concentration of non-target nucleic acid is at least 5-fold that of target nucleic acid in the heterogeneous sample, and wherein at least one of the assay samples comprises a single molecule of the target nucleic acid molecule;
- amplifying the single molecule of target nucleic acid to form an amplification product;
- hybridizing the amplification product to a first molecular beacon probe which hybridizes to the target nucleic acid and a second molecular beacon probe which hybridizes to the non-target nucleic acid, wherein each of the first and the second molecular beacon probes comprises a photoluminescent dye and a quenching agent at opposite 5′ and 3′ ends, wherein the photoluminescent dye on the first and second molecular beacon probes are different, and wherein the first molecular beacon probe further comprises a stem and a loop, wherein the stem comprises about 4 base pairs having a sequence 5′-CACG-3′, and the loop comprises about 16 base pairs and has a Tm of about 50-51° C.;
- and
- detecting the first and the second molecular beacon probes hybridized to the target nucleic acid, thereby detecting the target nucleic acid.
2 more elements
A method for detecting a target nucleic acid, the method comprising the steps of:
- separating a sample in which a target nucleic acid is present in an amount less than about 20% relative to non-target nucleic acid in said sample, to form a plurality of assay samples;
- amplifying said target nucleic acid in said assay samples;
- hybridizing the amplification products to a first molecular beacon probe which hybridizes to the target nucleic acid and to a second molecular beacon probe which hybridizes to the non-target nucleic acid, wherein each of the first and the second molecular beacon probes comprises a photoluminescent dye and a quenching agent at opposite 5′ and 3′ ends, wherein the photoluminescent dye on the first and second molecular beacon probes are different, and wherein the second molecular beacon probe further comprises a stem and a loop, wherein the stem comprises about 4 base pairs having a sequence 5′-CACG-3′, and the loop comprises about 19-20 base pairs and has a Tm of about 54-56° C.;
- and
- detecting the first and the second molecular beacon probes hybridized to the target nucleic acid, thereby detecting the target nucleic acid.
2 more elements
A method for detecting a target nucleic acid, the method comprising the steps of:
- providing a sample comprising X% of a target nucleic acid, wherein X is less than 100;
- dividing the sample to produce a plurality of assay samples;
- wherein the ratio of target to non-target nucleic acid in at least one of the samples is greater than X%;
- amplifying the target nucleic acid to form an amplification product;
- hybridizing the amplification products to a first molecular beacon probe which hybridizes to the target nucleic acid and a second molecular beacon probe which hybridizes to the non-target nucleic acid, wherein each of the first and the second molecular probes comprises a photoluminescent dye and a quenching agent at opposite 5′ and 3′ ends, wherein the photoluminescent dye on the first and second molecular beacon probes are different, and wherein the second molecular beacon probe further comprises a stem and a loop, wherein the stem comprises comprising about 4 base pairs having a sequence 5′-CACG-3′, and the loop comprises about 19-20 base pairs and a Tm of about 54-56° C.;
- and
- detecting the first and second molecular beacon probes hybridized to the target nucleic acid, thereby detecting the target nucleic acid.
4 more elements
A method for detecting a target nucleic acid in a population of non-target nucleic acid contained in a sample, the method comprising:
- dividing a heterogenous sample comprising target nucleic acid and non-target nucleic acid to form a plurality of assay samples, wherein the concentration of non-target nucleic acid is at least 5 fold that of target nucleic acid in the heterogeneous sample, and wherein at least one of the assay samples comprises a single molecule of the target nucleic acid;
- amplifying the single molecule of target nucleic acid to form an amplification product;
- hybridizing the amplification products to a first molecular beacon probe which hybridizes to the target nucleic acid and a second molecular beacon probe which hybridizes to the non-target nucleic acid, wherein each of the first and the second molecular probes comprises a photoluminescent dye and a quenching agent at opposite 5′ and 3′ ends, wherein the photoluminescent dye on the first and second molecular beacon probes are different, and wherein the second molecular beacon probe further comprises a stem and a loop, wherein the stem comprises about 4 base pairs having a sequence 5′-CACG-3′, and the loop comprises about 19-20 base pairs and a Tm of about 54-56° C.;
- and
- detecting the first and second molecular beacon probes hybridized to the target nucleic acid, thereby detecting the target nucleic acid.
2 more elements
Dependencies are inferred from the claim text.