Patent ID: 8642294
Filing Date: 2014-02-04
Classification: C12Q

Abstract:
1. A method for determining the pattern of cytosine methylation in a DNA specimen by comparing the amount of DNA fragments generated by a methylation-sensitive restriction enzyme with the amount of DNA fragments generated by a methylation-insensitive isoschizomer of the methylation-sensitive restriction enzyme, the method comprising: (a) digesting a first sample of the DNA specimen using the methylation-sensitive restriction enzyme to create double-stranded DNA fragments of ≦50 bp up to 2000 bp comprising regions of incomplete cytosine methylation in the DNA specimen; (b) annealing a plurality of pairs of single-stranded oligonucleotides to each other to form a plurality of double-stranded adaptors, wherein different pairs of the plurality of pairs of oligonucleotides have different nucleotide sequences than other pairs of the plurality of pairs of oligonucleotides; (c) ligating the double-stranded adaptors formed in step (b) to the ends of the double-stranded DNA fragments created in step (a) to form a first plurality of continuous nucleic acid sequences of the double-stranded DNA fragments flanked by adaptors on each end of the DNA fragments, wherein a first portion of the DNA fragments is flanked at each end by adaptors having identical nucleotide sequences and a second portion of the DNA fragments is flanked at each end by adaptors having non-identical nucleotide sequences; (d) quantifying the amount of DNA fragments resulting from step (c) of different lengths as a function of their location in the genome from which the DNA specimen was derived; (e) digesting a second sample of the DNA specimen using the methylation-insensitive isoschizomer of the methylation-sensitive restriction enzyme to create double-stranded DNA fragments of ≦50 bp up to 2000 bp representing the total potential repertoire of DNA fragments that would be created by the methylation-sensitive restriction enzyme in the absence of cytosine methylation at that restriction enzyme target site in the DNA specimen; (f) ligating double-stranded adaptors as formed in step (b) to the ends of the double-stranded DNA fragments created in step (e) to form a second plurality of continuous nucleic acid sequences of the double-stranded DNA fragments flanked by adaptors on each end of the DNA fragments, wherein a first portion of the DNA fragments is flanked at each end by adaptors having identical nucleotide sequences and a second portion of the DNA fragments is flanked at each end by adaptors having non-identical nucleotide sequences; (g) quantifying the amount of DNA fragments resulting from step (0 of different lengths as a function of their location in the genome from which the DNA specimen was derived, wherein the steps of quantifying the amounts of DNA fragments are carried out using polymerase chain reaction (PCR) amplification, wherein PCR amplification is carried out using a Mg 2+ concentration of 1-3 mM, (h) comparing the amount of the DNA fragments quantified in step (d) with the amount of DNA fragments quantified in step (g) so as to determine the pattern of cytosine methylation at target sites of the restriction enzymes in the DNA specimen, as a function of their location in the genome from which the DNA specimen was derived, wherein a greater amount of DNA fragments quantified in step (d) relative to step (g) indicates less cytosine methylation of the DNA fragments quantified in step (d).