SYSTEMS AND METHODS FOR METHYLATION PREDICTION
A method is provided for predicting an amount of methylation of at least one target region. The method includes establishing an observed size of a plurality of oligonucleotides relative to a size standard and correlating the observed size to a number of each of nucleotide base in each of the plurality of oligonucleotides. A mobility coefficient can be determined for each base of each respective oligonucleotide and the determined mobility coefficients can be applied to a predetermined number of polynucleotides subjected to methylation detection analysis. The plurality of oligonucleotides are treated with a modifying agent to obtain amplicons in methylated and unmethylated target regions and amplicons derived from methylated and unmethylated target regions are distinguished based on their relative mobilities. The degree of methylation can be predicted based on distinguished methylated regions.
1 . A method for predicting an amount of methylation of at least one target region, the method comprising:
establishing an observed size of a plurality of oligonucleotides relative to a size standard;
correlating the observed size to a number of each of nucleotide base in each of the plurality of oligonucleotides;
determining a mobility coefficient for each base of each respective oligonucleotide;
applying determined mobility coefficients to a predetermined number of polynucleotides subjected to methylation detection analysis;
treating said plurality of oligonucleotides with a modifying agent to obtain amplicons in methylated and unmethylated target regions;
distinguishing amplicons derived from methylated and unmethylated target regions based on their relative mobilities; and
predicting the degree of methylation of distinguished methylated regions.
2 . The method of claim 1 , further comprising calculating a predicted size of each of the predetermined number of subjected polynucleotides.
3 . The method of claim 2 , wherein calculating the predicted size includes using a known sequence of the amplicon and presumed sequence of the amplicon arising from a per-methylated dDNA, and calculating the predicted size for each of the two products.
4 . The method of claim 1 , wherein establishing an observed size of a plurality of oligonucleotides relative to a size standard includes providing a predetermined panel of oligonucleotides as a learning data set, and measuring a size of each of the plurality of oligonucleotides by capillary electrophoresis using the size standard.
5 . The method of claim 1 , wherein the observed size is related to a length of a corresponding oligonucleotide.
6 . The method of claim 1 , wherein the observed size is related to a composition of a corresponding oligonucleotide.
7 . The method of claim 1 , wherein correlating the observed size to the number of each of the nucleotide bases in each of the plurality of oligonucleotides includes at least the equation: size=a A+g G+t T+c C, where A, G, T and C are the numbers of nucleotides present in a single strand DNA, and a, g, t, and c are base-specific mobility coefficients.
8 . The method of claim 7 , wherein observed size for a set of at least five oligonucleotides with known base compositions enables calculation of the base-specific mobility coefficients under predetermined separation conditions, where the coefficients are used to calculate the predicted size of any oligonucleotide having the known base composition under the same separation conditions.
9 . The method of claim 4 , wherein determining coefficients of oligonucleotide compositions from the learning data set comprises:
performing a regression analysis on the data set using length of oligonucleotide; and
performing a regression analysis on the data set using composition of oligonucleotide.
10 . The method of claim 4 , wherein the learning set includes a set of 50 synthetic oligonucleotides from about 19 to about 61 nts.
11 . The method of claim 1 , wherein the modifying agent includes sodium bisulfite.
12 . A method for predicting a size of amplicons generated from methylated and unmethylated gDNA, the method comprising:
establishing an observed size of a plurality of oligonucleotides relative to a size standard;
correlating the observed size to a number of each of the nucleotide bases in each of the plurality of oligonucleotides;
determining a mobility coefficient for each base of each respective oligonucleotide;
applying determined mobility coefficients to a predetermined number of polynucleotides subjected to methylation detection analysis; and
calculating the predicted size of the amplicons using the determined mobility coefficients and a known sequence of the amplicon and a presumed sequence of an amplicon arising from a per-methylated gDNA.
13 . The method of claim 12 , wherein establishing an observed size of a plurality of oligonucleotides relative to a size standard includes providing a predetermined panel of oligonucleotides as a learning data set, and measuring a size of each of the plurality of oligonucleotides by capillary electrophoresis using the size standard.
14 . The method of claim 12 , wherein the observed size is related to a length of a corresponding oligonucleotide.
15 . The method of claim 12 , wherein the observed size is related to a composition of a corresponding oligonucleotide.
16 . The method of claim 12 , wherein correlating the observed size to the number of each of the nucleotide bases in each of the plurality of oligonucleotides includes at least the equation: size=a A+g G+t T+c C, where A, G, T and C are the numbers of nucleotides present in a single strand DNA, and a, g, t, and c are base-specific mobility coefficients.
17 . The method of claim 16 , wherein observed size for a set of at least five oligonucleotides with known base compositions enables calculation of the base-specific mobility coefficients under predetermined separation conditions, where the coefficients are used to calculate the predicted size of any oligonucleotide having the known base composition under the same separation conditions.
18 . The method of claim 13 , wherein determining coefficients of oligonucleotide compositions from the learning data set comprises:
performing a regression analysis on the data set using length of oligonucleotide; and
performing a regression analysis on the data set using composition of oligonucleotide.
19 . The method of claim 13 , wherein the learning set includes a set of 50 synthetic oligonucleotides from about 19 to about 61 nts.
20 . The method of claim 12 , wherein the modifying agent includes sodium bisulfite.
21 . A method of calculating a predicted size for an untreated (DNA) product and a bisulfite treated (DNA) product comprising:
providing a known sequence of an amplicon and a presumed sequence of an amplicon arising from a per-methylated gDNA;
calculating a DNA fragment size to a length of the corresponding oligonucleotide; and
calculating a DNA fragment size to a composition of the corresponding oligonucleotide.