Methods and compositions for generating and amplifying DNA libraries for sensitive detection and analysis of DNA methylation
First Claim
1. A method of preparing a DNA molecule, comprising:
- (a) providing a DNA molecule;
(b) digesting the DNA molecule with at least one methylation-sensitive restriction enzyme and, optionally, an additional nuclease, to provide digested DNA molecules;
(c) incorporating a nucleic acid molecule into at least some of the digested DNA molecules to provide first modified DNA molecules, by;
incorporating at least one primer from a plurality of primers, said primers comprising a 5′
constant sequence and a 3′
variable sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein the sequence of the constant and variable regions consists essentially of only two types of non-complementary nucleotides selected from the group consisting of adenines and guanines;
adenines and cytosines;
guanines and thymidines; and
cytosines and thymidines, such that the primers of the population will not cross-hybridize or self-hybridize under conditions employed in step d); and
(d) amplifying one or more of the first modified DNA molecules to provide amplified first modified DNA molecules.
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Accused Products
Abstract
The present invention regards a variety of methods and compositions for obtaining epigenetic information, such as DNA methylation patterns, through the preparation, amplification and analysis of Methylome libraries. In particular, the method employs preparation of a DNA molecule by digesting the DNA molecule with at least one methylation-sensitive restriction enzyme; incorporating a nucleic acid molecule into at least some of the digested DNA molecules by either (1) incorporating at least one primer from a plurality of primers that have a 5′ constant sequence and a 3′ variable sequence, wherein the primers are substantially non-self-complementary and substantially non-complementary to other primers in the plurality; or (2) incorporating an oligonucleotide having an inverted repeat and a loop under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the digested DNA molecule, followed by polymerization from a 3′ hydroxyl group present in a nick in the oligonucleotide-linked molecule; and amplifying one or more of the DNA molecules.
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Citations
37 Claims
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1. A method of preparing a DNA molecule, comprising:
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(a) providing a DNA molecule; (b) digesting the DNA molecule with at least one methylation-sensitive restriction enzyme and, optionally, an additional nuclease, to provide digested DNA molecules; (c) incorporating a nucleic acid molecule into at least some of the digested DNA molecules to provide first modified DNA molecules, by; incorporating at least one primer from a plurality of primers, said primers comprising a 5′
constant sequence and a 3′
variable sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein the sequence of the constant and variable regions consists essentially of only two types of non-complementary nucleotides selected from the group consisting of adenines and guanines;
adenines and cytosines;
guanines and thymidines; and
cytosines and thymidines, such that the primers of the population will not cross-hybridize or self-hybridize under conditions employed in step d); and(d) amplifying one or more of the first modified DNA molecules to provide amplified first modified DNA molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method of preparing a DNA molecule, comprising:
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(a) providing a DNA molecule; (b) digesting the molecule with one or more methylation-specific endonucleases to provide DNA fragments; (c) incorporating a nucleic acid molecule into the DNA fragments to provide first modified DNA molecules, by a method comprising; incorporating at least one primer from a plurality of primers, said primer comprising a 5′
constant sequence and a 3′
variable sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, and further wherein the sequence of the constant and variable regions consists essentially of only two types of non-complementary nucleotides selected from the group consisting of adenines and guanines;
adenines and cytosines;
guanines and thymidines; and
cytosines and thymidines, such that the primers of the population will not cross-hybridize or self-hybridize under conditions employed in step d); and(d) amplifying at least one first modified DNA molecule to provide amplified DNA molecules. - View Dependent Claims (27, 28, 29, 30)
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31. A method of preparing a DNA molecule, comprising:
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(a) providing a bisulfite-converted DNA molecule; (b) incorporating a nucleic acid molecule into at least some of the digested DNA molecules to provide first modified DNA molecules, by incorporating at least one primer from a plurality of primers, said primers comprising a 5′
constant sequence and a 3′
variable sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein the sequence of the constant and variable regions consists essentially of only two types of non-complementary nucleotides selected from the group consisting of adenines and guanines;
adenines and cytosines;
guanines and thymidines; and
cytosines and thymidines, such that the primers of the population will not cross-hybridize or self-hybridize under conditions employed in step d); and(c) amplifying one or more of the first modified DNA molecules to provide amplified first modified DNA molecules. - View Dependent Claims (32, 33, 34, 35, 36, 37)
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Specification