Isolation of CpG islands by thermal segregation and enzymatic selection-amplification method
First Claim
1. A method of amplifying a plurality of amplifiable DNA molecules, comprising:
- providing a plurality of DNA molecules, said plurality having molecules comprising one or more regions that are GC-poor and having molecules comprising one or more regions that are GC-rich;
subjecting the plurality of DNA molecules to sufficient conditions to denature GC-poor regions but not to denature GC-rich regions, thereby producing GC-rich regions suitable for amplification; and
subjecting the plurality to amplification conditions such that the denatured GC-poor regions are substantially not amplified and such that one or more of the non-denatured or partially denatured GC-rich regions are amplified.
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Abstract
The present invention concerns isolation, library preparation and selective amplification from a compositionally heterogeneous pool of DNA fragments of a fraction of molecules, such as those originating from promoter CpG islands and characterized by a high GC content. In particular, the process utilizes a heat-induced segregation of DNA molecules into GC-poor, single-stranded molecule fractions and GC-rich, double-stranded molecule fractions, with subsequent enzymatic conversion of the GC-rich, double-stranded DNA molecules into a library, and, optionally, amplification. In specific embodiments, the isolation process is used to generate promoter-enriched genomic and methylome libraries for research and diagnostic applications, for example.
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Citations
35 Claims
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1. A method of amplifying a plurality of amplifiable DNA molecules, comprising:
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providing a plurality of DNA molecules, said plurality having molecules comprising one or more regions that are GC-poor and having molecules comprising one or more regions that are GC-rich;
subjecting the plurality of DNA molecules to sufficient conditions to denature GC-poor regions but not to denature GC-rich regions, thereby producing GC-rich regions suitable for amplification; and
subjecting the plurality to amplification conditions such that the denatured GC-poor regions are substantially not amplified and such that one or more of the non-denatured or partially denatured GC-rich regions are amplified. - 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)
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29. A method of amplifying a CpG island from a molecule, comprising:
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providing a plurality of DNA molecules, said plurality comprising at least one molecule having at least one CpG island and said plurality having at least one molecule comprising one or more regions that are GC-poor;
subjecting the plurality of DNA molecules to sufficient conditions to denature GC-poor regions but not to substantially denature the CpG island, thereby rendering the CpG island suitable for amplification; and
subjecting the plurality to amplification conditions such that the denatured GC-poor regions are substantially not amplified and such that the non-denatured or partially denatured CpG-island is amplified. - View Dependent Claims (30)
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31. A method of preparing a library, comprising:
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providing a plurality of DNA molecules, said plurality having molecules comprising one or more regions that are GC-poor and having molecules comprising one or more regions that are GC-rich;
subjecting the plurality of DNA molecules to sufficient conditions to denature GC-poor regions but not to denature GC-rich regions, thereby producing GC-rich regions suitable for amplification; and
subjecting the plurality to amplification conditions such that the denatured GC-poor regions are substantially not amplified and such that one or more of the non-denatured or partially denatured GC-rich regions are amplified. - View Dependent Claims (32, 33, 34, 35)
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Specification