System and method for temperature gradient capillary electrophoresis
First Claim
1. A temperature gradient electrophoresis-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample having non-desalted polymerase chain reaction (PCR) products, comprising:
- providing a first nucleotide-containing compound having non-desalted polymerase chain reaction (PCR) products and a second nucleotide-containing compound;
subjecting the first and second nucleotide-containing compounds to temperature gradient gel electrophoresis to cause the first and second nucleotide-containing compounds to migrate at different relative rates;
irradiating the non-desalted polymerase chain reaction (PCR) products within the first nucleotide-containing compound to generate a first spectroscopic signal, and irradiating the second nucleotide-containing compound to generate a second spectroscopic signal; and
converting the first and second spectroscopic signals into first and second data suitable for determining the presence of a single nucleotide polymorphism or a mutation in the first nucleotide-containing compound.
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Abstract
The present invention relates to a method for determining the presence of a mutation in a first sample comprising first nucleotides. The reference sample comprising reference nucleotides. The first sample and a reference sample are subjected to electrophoresis in the presence of at least one intercalating dye. During electrophoresis the temperature of the first sample and the reference sample is changed by an amount sufficient to change an electrophoretic mobility of at least one of the first or reference nucleotides. Fluorescence intensity data are obtained. The fluorescence intensity data are indicative of the presence of the first and reference nucleotides. At least one of the first sample or reference samples comprises products resulting from a polymerase chain reaction (PCR), the products not having been desalted prior to electrophoresis.
18 Citations
21 Claims
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1. A temperature gradient electrophoresis-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample having non-desalted polymerase chain reaction (PCR) products, comprising:
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providing a first nucleotide-containing compound having non-desalted polymerase chain reaction (PCR) products and a second nucleotide-containing compound;
subjecting the first and second nucleotide-containing compounds to temperature gradient gel electrophoresis to cause the first and second nucleotide-containing compounds to migrate at different relative rates;
irradiating the non-desalted polymerase chain reaction (PCR) products within the first nucleotide-containing compound to generate a first spectroscopic signal, and irradiating the second nucleotide-containing compound to generate a second spectroscopic signal; and
converting the first and second spectroscopic signals into first and second data suitable for determining the presence of a single nucleotide polymorphism or a mutation in the first nucleotide-containing compound. - View Dependent Claims (2, 3)
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4. A method for determining the presence of a single nucleotide polymorphism or a mutation in a biological sample having non-desalted polymerase chain reaction (PCR) products, comprising:
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obtaining a first parameter representative of a first spectroscopic signal resulting from irradiating a first nucleotide-containing compound having non-desalted polymerase chain reaction (PCR) products, the first nucleotide-containing compound having been subjected to temperature gradient gel electrophoresis;
obtaining a second parameter representative of a second spectroscopic signal resulting from irradiating a second nucleotide-containing compound, the second nucleotide-containing compound having been subjected to temperature gradient electrophoresis;
comparing the first and second parameters to determine whether there is a single nucleotide polymorphism or a mutation in the first nucleotide-containing compound. - View Dependent Claims (5, 6)
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7. A temperature gradient electrophoresis-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample having single-stranded nucleic acid PCR products, comprising:
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providing a first nucleotide-containing compound having single-stranded nucleic acid PCR products and a second nucleotide-containing compound;
subjecting the first and second nucleotide-containing compounds to temperature gradient electrophoresis to cause the first and second nucleotide-containing compounds to migrate at different relative rates;
irradiating the first nucleotide-containing compound having single stranded nucleic acid PCR products to generate a first spectroscopic signal, and irradiating the second nucleotide-containing compound to generate a second spectroscopic signal; and
converting the first and second spectroscopic signals into first and second data suitable for determining the presence of a single nucleotide polymorphism or a mutation in the first nucleotide-containing compound.
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8. A method for determining the presence of a single nucleotide polymorphism or a mutation in a biological sample having single-stranded nucleic acid PCR products, comprising:
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obtaining a first parameter representative of a first spectroscopic time signal resulting from irradiating a first nucleotide-containing compound having single-stranded nucleic acid PCR products, the first nucleotide-containing compound having been subjected to temperature gradient electrophoresis;
obtaining a second parameter representative of a second spectroscopic time signal resulting from irradiating a second nucleotide-containing compound that has been subjected to temperature gradient electrophoresis;
comparing the first and second parameters to determine whether there is a single nucleotide polymorphism or a mutation in the first nucleotide-containing compound.
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9. A temperature gradient electrophoresis-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample comprising first and second pairs of nucleotide-containing compounds, each pair of nucleotide-containing compounds comprising first and second members, comprising:
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while subjecting the sample to electrophoresis, changing the temperature at a first rate for a first period of time by an amount sufficient to change an electrophoretic mobility of the first member of the first pair of nucleotide-containing compounds relative to an electrophoretic mobility of the second member of the first pair of nucleotide-containing compounds and changing the temperature at a second, different rate for a second period of time by an amount sufficient to change an electrophoretic mobility of the first member of the second pair of nucleotide-containing compounds relative an electrophoretic mobility of the second member of the second pair of nucleotide-containing compounds; and
irradiating the first pair of nucleotide-containing compounds to generate a first spectroscopic signals, and irradiating the second nucleotide-containing compounds to generate second spectroscopic signals; and
converting the first and second spectroscopic signals into first and second data suitable for determining the presence of a single nucleotide polymorphism or a mutation.
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10. A method for determining the presence of a single nucleotide polymorphism or a mutation in a biological sample comprising first and second pairs of nucleotide containing compounds, comprising:
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obtaining a first parameter representative of a first respective pair of spectroscopic signals resulting from irradiating the first pair of nucleotide-containing compounds, the first pair of nucleotide-containing compounds having been subjected to temperature gradient electrophoresis by changing a temperature at a first rate during the electrophoresis;
obtaining a second parameter representative of a second respective pair of spectroscopic signals resulting from irradiating the second pair of nucleotide-containing compounds, the second pair of nucleotide-containing compounds having been been subjected to temperature gradient electrophoresis by changing a temperature at a second, different rate during the electrophoresis;
comparing the first and second parameters with a reference parameter to determine whether there is a single nucleotide polymorphism or a mutation in the first or second pairs of nucleotide-containing compounds.
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11. A temperature gradient electrophoresis-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample, comprising:
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providing first and second nucleotide-containing compounds;
simultaneously subjecting the first and second nucleotide-containing compounds to temperature gradient electrophoresis to cause the first and second nucleotide-containing compounds to migrate at different relative rates;
irradiating the first nucleotide-containing compound to generate a first spectroscopic signal having a first wavelength, and irradiating the second nucleotide-containing compound to generate a second spectroscopic signal having a second, different wavelength; and
converting the first and second spectroscopic signals into first and second data suitable for determining the presence of a single nucleotide polymorphism or a mutation in at least one of the first and second nucleotide-containing compounds.
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12. A method for determining the presence of a single nucleotide polymorphism or a mutation in a biological sample, comprising:
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obtaining a first parameter representative of a first spectroscopic signal resulting from irradiating a first nucleotide-containing compound, the first nucleotide-containing compound having been subjected to temperature gradient electrophoresis, the first spectroscopic signal having a first wavelength;
obtaining a second parameter representative of a second spectroscopic signal resulting from irradiating a second nucleotide-containing compound, the second nucleotide-containing compound having been subjected to temperature gradient electrophoresis, the second spectroscopic signal having a second, different wavelength;
comparing the first and second parameters with a reference parameter to determine whether there is a single nucleotide polymorphism or a mutation in at least one of the first and second nucleotide-containing compounds.
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13. A capillary-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample comprising first and second pairs of nucleotide-containing compounds, member compounds of the first pair having different sizes than member compounds of the second pair, the method comprising:
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subjecting the first and second pairs of nucleotide-containing compounds to electrophoresis;
during electrophoresis, changing the temperature of a central portion of the capillary from a first temperature T1 to a second temperature T2, the temperature being changed at a rate r given by where t2 is the migration time required for the first pair of nucleotide-containing compounds to exit the central portion when the capillary is at temperature T1 and t1 is the migration time required for the second pair of nucleotide containing compounds to enter the central portion when the capillary is at a temperature T2;
irradiating the first pair of nucleotide-containing compound to generate first spectroscopic signals, and irradiating the second pair of nucleotide-containing compounds to generate a second pair of spectroscopic signals; and
converting the first and second pairs of spectroscopic signals into first and second data suitable for determining the presence of a single nucleotide polymorphism in at least one of the members of the first and second pairs of nucleotide containing compounds.
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14. A temperature gradient electrophoresis-based method for generating data indicative of the presence of a single nucleotide polymorphism or a mutation in a biological sample, comprising:
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subjecting the first nucleotide-containing compound to electrophoresis in a channel disposed in a substantially planar substrate;
during electrophoresis, changing a temperature of the first nucleotide-containing compound by an amount sufficient to change an electrophoretic mobility of the first nucleotide-containing compound;
thermo-electrically reducing the temperature of the first nucleotide-containing compound;
irradiating the first nucleotide-containing compound to generate a first spectroscopic signal; and
converting the first spectroscopic signal into first data suitable for determining the presence of a single nucleotide polymorphism or a mutation in the first nucleotide-containing compound.
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15. In a method for detecting mutations in a nucleotide-containing sample by subjecting nucleotide containing sample components to temperature gradient electrophoresis and obtaining spectroscopic intensity data indicative of the presence of the nucleotides, the improvement comprising:
actively reducing the temperature of the nucleotide-containing sample components after the nucleotides have been subjected to temperature gradient electrophoresis and prior to obtaining the spectroscopic intensity data. - View Dependent Claims (16, 17, 18, 19, 20, 21)
Specification