System and method for determining the presence of methylated cytosines in polynucleotides
First Claim
1. A temperature gradient electrophoresis-based method for generating data indicative of the presence of one or more methylated cytosines in a sample comprising a first nucleotide containing compound (NCC) having non-methylated cytosines, comprising:
- contacting a first portion of the sample comprising the first NCC with a first compound to thereby provide a modified NCC, wherein non-methylated cytosines of the first NCC are replaced with a different base;
amplifying the modified NCC to obtain first PCR products;
preparing, from a second portion of the sample comprising the first NCC, a reference NCC having a sequence that differs from a sequence of the first PCR products at locations corresponding to the presence of the methylated cytosines in the first NCC;
subjecting the first PCR products to temperature gradient electrophoresis in the presence of the reference NCC;
irradiating the first PCR products and reference NCC with light to thereby generate a spectroscopic signal; and
converting the spectroscopic signal into data indicative of the presence of the one or more methylated cytosines in the first NCC.
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Abstract
The present invention relates to a method for determining the presence of a methylated cytosine in a first sample comprising a first nucleotide containing compound. The first sample and a reference NCC are subjected to electrophoresis in the presence of at least one intercalating dye. During electrophoresis the temperature of the first NCC and the reference NCC is changed by an amount sufficient to change an electrophoretic mobility of at least one of the first or reference NCCs. Fluorescence intensity data are obtained. The fluorescence intensity data are indicative of the presence of the first and reference NCCs.
20 Citations
20 Claims
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1. A temperature gradient electrophoresis-based method for generating data indicative of the presence of one or more methylated cytosines in a sample comprising a first nucleotide containing compound (NCC) having non-methylated cytosines, comprising:
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contacting a first portion of the sample comprising the first NCC with a first compound to thereby provide a modified NCC, wherein non-methylated cytosines of the first NCC are replaced with a different base;
amplifying the modified NCC to obtain first PCR products;
preparing, from a second portion of the sample comprising the first NCC, a reference NCC having a sequence that differs from a sequence of the first PCR products at locations corresponding to the presence of the methylated cytosines in the first NCC;
subjecting the first PCR products to temperature gradient electrophoresis in the presence of the reference NCC;
irradiating the first PCR products and reference NCC with light to thereby generate a spectroscopic signal; and
converting the spectroscopic signal into data indicative of the presence of the one or more methylated cytosines in the first NCC. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A temperature gradient electrophoresis-based method for generating data indicative of the presence of one or more methylated cytosines in a sample comprising a first nucleotide containing compound (NCC), comprising:
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obtaining first PCR products formed by;
(a1) contacting the first NCC with a first compound to thereby provide a modified NCC in which non-methylated cytosines are replaced with a different base, and (a2) amplifying the modified NCC to obtain said first PCR products;
obtaining a reference NCC, the reference NCC being prepared from a second portion of the sample comprising the first NCC, the reference NCC having a sequence that differs from a sequence of the first PCR products at locations corresponding to the presence of the methylated cytosines in the first NCC;
subjecting the first PCR products and a reference NCC to temperature gradient electrophoresis, irradiating the first PCR products and reference NCC with light to thereby generate a spectroscopic signal; and
converting the spectroscopic signal into data indicative of the presence of the one or more methylated cytosines in the first NCC. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification