METHOD FOR MEASURING DNA METHYLATION PROFILES
Methods are provided for determining epimutations in a nucleic acid sequence of a cell.
1 . A method for determining a pattern of epimutations in a nucleic acid sequence comprising:
optionally, mixing the nucleic acid with an amount of a heterologous nucleic acid;
subjecting the nucleic acid to nucleic acid-denaturing conditions;
contacting the nucleic acid with sodium bisulfite at a temperature in excess of 50° C. so as to convert non-methylated cytosine residues of the nucleic acid into uracil residues;
recovering the converted nucleic acid;
subjecting the recovered converted nucleic acid to amplification with primers directed to the nucleic acid sequence;
sequencing the amplified product to determine the presence of uracil residues and cytosine residues, so as to thereby determine the pattern of epimutations in the nucleic acid sequence, wherein a cytosine residue in the amplified product indicates a methylated cytosine at the corresponding residue position in the nucleic acid sequence and wherein a uracil residue in the amplified product indicates a non-methylated cytosine at the corresponding residue position in the nucleic acid sequence.
2 . The method of claim 1 , further comprising comparing the epimutation pattern of the amplified product to a corresponding control sequence, so as to determine epimutations in the nucleic acid sequence relative to the control sequence.
3 . The method of claim 1 , wherein the nucleic acid is obtained from a cell.
4 . The method of claim 1 , further comprising obtaining the nucleic acid from a single cell.
5 . The method of claim 1 , wherein the heterologous nucleic acid is DNA or tRNA.
6 . The method of claim 1 , wherein the heterologous nucleic acid is a salmon sperm DNA or salmon sperm tRNA.
7 . The method of claim 1 , wherein the amplification primers are not directed to the heterologous nucleic acid.
8 . (canceled)
9 . The method of claim 1 , wherein the nucleic acid is contacted with sodium bisulfite at a temperature of between 60° C. and 70° C. so as to convert methylated cytosine residues of the nucleic acid into uracil residues.
10 . The method of claim 1 , wherein the nucleic acid is contacted with sodium bisulfite at a temperature and for an amount of time sufficient as to convert 95% or more of the methylated cytosine residues of the nucleic acid into uracil residues.
11 . The method of claim 1 , further comprising contacting the nucleic acid, subsequent to the contact with sodium bisulfite, with carrier RNA.
12 - 14 . (canceled)
15 . The method of claim 1 , wherein the method is performed on nucleic acid obtained from a single cell.
16 - 18 . (canceled)
19 . The method of claim 1 , wherein the recovered nucleic acid is subjected to whole genome amplification using random hexamer primers.
20 . The method of claim 1 , wherein the amplification is multiple displacement amplification.
21 - 26 . (canceled)
27 . The method claim 1 , wherein the recovered nucleic acid is subjected to nested PCR amplification prior to sequencing.
28 . The method of claim 1 , wherein recovering the converted nucleic acid comprises purifying the converted nucleic acid from remaining reagents and products using a nucleic acid purification column.
29 . The method of claim 1 , further comprising desulphonating the recovered nucleic acid.
30 . A method for determining the effect of an agent on epimutation status of a nucleic acid comprising determining the epimutation pattern of a nucleic acid in the cells in a first portion of the cells, and contacting a second portion of the cells with the agent and determining epimutation pattern in a corresponding nucleic acid sequence in the second portion of the cells by the method of claim 1 , and comparing the pattern of epimutations in the nucleic acid obtained from the first portion with the pattern of epimutations in the corresponding nucleic acid from the second portion so as to determine the effect of the agent on epimutation status of the cells.
31 . (canceled)
32 . A kit for performing the method of claim 1 comprising reagents as set forth in claim 1 and written instructions for use.
33 . A system for performing the method of claim 1 , comprising:
one or more data processing apparatus; and
a computer-readable medium coupled to the one or more data processing apparatus having instructions stored thereon which, when executed by the one or more data processing apparatus, cause the one or more data processing apparatus to perform the method of claim 1 .
34 . A computer-readable medium coupled to the one or more data processing apparatus having instructions stored thereon which, when executed by the one or more data processing apparatus, cause the one or more data processing apparatus to perform the method of claim 1 .