Methods and compositions for generating and amplifying DNA libraries for sensitive detection and analysis of DNA methylation
First Claim
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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 one of the following;
(1) 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;
or (2) incorporating an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the molecule, thereby producing an oligonucleotide-linked molecule comprising a nick having a 3′
hydroxyl group, wherein there is polymerization from the 3′
hydroxyl group of at least part of the oligonucleotide-linked molecule; and
(d) amplifying one or more of the first modified DNA molecules to provide amplified first modified DNA molecules.
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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 several aspects of the present invention, there are methods based on methylation-dependent enrichment or depletion of genomic DNA isolated from cellular and cell-free sources. In additional embodiments, there are methods and compositions for single-step high throughput preparations of Methylome libraries.
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Citations
73 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 one of the following;
(1) 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;
or(2) incorporating an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the molecule, thereby producing an oligonucleotide-linked molecule comprising a nick having a 3′
hydroxyl group, wherein there is polymerization from the 3′
hydroxyl group of at least part of the oligonucleotide-linked molecule; 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. 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 restriction enzymes to provide DNA fragments;
(c) incorporating a nucleic acid molecule into the DNA fragments to provide first modified DNA molecules, by a method comprising;
(1) 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;
(2) incorporating a first adaptor having a nonblocked 3′
end to produce first adaptor-linked molecules, wherein the 5′
end of the fragment is attached to the nonblocked 3′
end of the adaptor, leaving a nick site between the juxtaposed 3′
end of the fragment and a 5′
end of the first adaptor, and polymerizing the 3′
end of the fragment from the nick site;
or(3) incorporating an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the fragment, thereby producing an oligonucleotide-linked molecule comprising a nick having a 3′
hydroxyl group, wherein there is polymerization from the 3′
hydroxyl group of at least part of the oligonucleotide-linked molecule; and
(d) amplifying at least one first modified DNA molecule to provide amplified DNA molecules. - View Dependent Claims (41, 42, 43)
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44. A method of preparing a DNA molecule, comprising:
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(a) providing one or more nucleic acid molecules;
(b) incorporating a nucleic acid molecule into the molecules by one or more of the following, wherein the incorporated molecule is resistant to bisulfite conversion, to provide first modified DNA molecules;
(1) incorporating sequence by attaching a first adaptor having a nonblocked 3′
end to the ends of the molecule to produce first adaptor-linked molecules, wherein the 5′
end of the molecule is attached to the nonblocked 3′
end of the adaptor, leaving a nick site between the juxtaposed 3′
end of the molecule and a 5′
end of the first adaptor, and extending the 3′
end of the molecule from the nick site;
or(2) incorporating sequence by providing an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the DNA molecule, thereby producing an oligonucleotide-linked DNA molecule comprising a nick having a 3′
hydroxyl group, wherein there is polymerization from the 3′
hydroxyl group of at least part of the oligonucleotide-linked DNA molecule;
(c) providing sodium bisulfite to said first modified nucleic acid molecules, wherein the unmethylated cytosines in said nucleic acid molecules are converted to uracil, thereby producing bisulfite-converted single-stranded nucleic acid molecules; and
(d) amplifying one or more of the bisulfite-converted molecules. - View Dependent Claims (45, 46, 47, 48)
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49. A method of preparing a DNA molecule, comprising the steps of:
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(1) providing a DNA molecule;
(2) altering the molecule in a single incubation to produce oligonucleotide-linked molecules, said incubation comprising two of more of the following;
(a) modifying the ends of the DNA molecules to provide attachable ends;
(b) repairing nicks and/or gaps within the DNA molecules;
(c) attaching a first oligonucleotide comprising a known sequence and a nonblocked 3′
end to the ends of the DNA molecules to produce oligonucleotide-linked molecules, wherein the 5′
end of the DNA is attached to the nonblocked 3′
end of the oligonucleotide, leaving a nick site between the juxtaposed 3′
end of the DNA molecule and a 5′
end of the oligonucleotide; and
(d) polymerizing from the 3′
end of the molecule from the nick site;
(3) digesting the oligonucleotide-linked DNA molecules with a mixture of methylation-sensitive restriction enzymes that do not cleave within the attached first oligonucleotide; and
(4) amplifying the digested first oligonucleotide-linked DNA molecules with a primer complementary to at least a portion of the stem region of the oligonucleotide to produce amplified oligonucleotide-linked fragments. - View Dependent Claims (50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72)
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73. A method of preparing a DNA molecule, comprising:
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(a) providing a DNA molecule resulting from apoptotic degradation;
(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 an adaptor having a nonblocked 3′
end to at least some of the digested DNA molecules to produce adaptor-linked molecules, wherein the 5′
end of the molecule is attached to the nonblocked 3′
end of the adaptor, leaving a nick site between the juxtaposed 3′
end of the molecule and a 5′
end of the adaptor, and polymerizing from the 3′
end of the molecule from the nick site, to produce a modified DNA molecule; and
(d) amplifying one or more of the modified DNA molecules to provide amplified modified DNA molecules.
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Specification