System and method for determining known DNA variants with temperature gradient electrophoresis
First Claim
1. A method of determining the genotype of a sample polynucleotide having at least a first variant site, comprising:
- amplifying at least a portion of the sample polynucleotide to obtain first amplicons, the first amplicons including the first variant site;
combining the first amplicons with first and second different polynucleotide controls, the first and second polynucleotide controls differing by at least one base therealong, the position of the at least one differing base corresponding to the first variant site of the sample polynucleotide;
preparing a plurality of first duplexes, each of at least some of the first duplexes comprising (i) a polynucleotide strand of one of the first amplicons and (ii) a complementary polynucleotide strand of the first polynucleotide control;
preparing a plurality of second duplexes, each of at least some of the second duplexes comprising (i) a polynucleotide strand of one of the first amplicons and (ii) a complementary polynucleotide strand of the second polynucleotide control;
subjecting the first and second duplexes to temperature gradient electrophoresis (TGE) to obtain first and second electrophoresis data; and
determining the genotype of the first variant site of the sample polynucleotide based on the first and second electrophoresis data.
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Abstract
The present invention relates to a method of determining the genotype of a sample polynucleotide having at least a first variant site. At least a portion of the sample polynucleotide is amplified to obtain first amplicons, the first amplicons including the first variant site. The first amplicons are combined with first and second different polynucleotide controls, the first and second polynucleotide controls differing by at least one base therealong, the position of the at least one differing base corresponding to the first variant site of the sample polynucleotide. A plurality of first duplexes are prepared, each of at least some of the first duplexes comprising (i) a polynucleotide strand of one of the first amplicons and (ii) a complementary polynucleotide strand of the first polynucleotide control. A plurality of second duplexes are prepared, each of at least some of the second duplexes comprising (i) a polynucleotide strand of one of the first amplicons and (ii) a complementary polynucleotide strand of the second polynucleotide control. The first and second duplexes are subjected to temperature gradient electrophoresis (TGE) to obtain first and second electrophoresis data. The genotype of the first variant site of the sample polynucleotide is determiend based on the first and second electrophoresis data.
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Citations
14 Claims
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1. A method of determining the genotype of a sample polynucleotide having at least a first variant site, comprising:
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amplifying at least a portion of the sample polynucleotide to obtain first amplicons, the first amplicons including the first variant site; combining the first amplicons with first and second different polynucleotide controls, the first and second polynucleotide controls differing by at least one base therealong, the position of the at least one differing base corresponding to the first variant site of the sample polynucleotide; preparing a plurality of first duplexes, each of at least some of the first duplexes comprising (i) a polynucleotide strand of one of the first amplicons and (ii) a complementary polynucleotide strand of the first polynucleotide control; preparing a plurality of second duplexes, each of at least some of the second duplexes comprising (i) a polynucleotide strand of one of the first amplicons and (ii) a complementary polynucleotide strand of the second polynucleotide control; subjecting the first and second duplexes to temperature gradient electrophoresis (TGE) to obtain first and second electrophoresis data; and determining the genotype of the first variant site of the sample polynucleotide based on the first and second electrophoresis data. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method for determining the genotype of a sample polynucleotide, compnsing:
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providing first and second polynucleotide controls, the first and second polynucleotide controls differing by at least one base therealong, the position of the differing base corresponding to a position of a variant site of the sample polynucleotide; combining a first amount of the sample polynucleotide with the first polynucleotide control to prepare a first mixture, each of the sample polynucleotide and the first polynucleotide control comprising a polynucleotide strand sufficiently complementary to form a duplex with a polynucleotide strand of the other of the sample polynucleotide and first polynucleotide control; forming first duplexes, at least some of the first duplexes comprising a strand of the sample polynucleotide and a strand of the first polynucleotide control; combining a first amount of the sample polynucleotide with the second polynucleotide control to prepare a second mixture, each of the sample polynucleotide and the second polynucleotide control comprising a polynucleotide strand sufficiently complementary to form a duplex with a polynucleotide strand of the other of the sample polynucleotide and second polynucleotide control; subjecting the first and second mixtures to temperature gradient electrophoresis to obtain first and second electrophoresis data; and determining the genotype of the sample polynucleotide based on the first and second electrophoresis data. - View Dependent Claims (12, 13, 14)
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Specification