Methods for the amplification of bisulfite-treated DNA
Inventors
He, Chuan • NIE, Ji • CUI, Xiao-Long
Assignees
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Publication Number
US-12606866-B2
Publication Date
2026-04-21
Expiration Date
Abstract
The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.
Core Innovation
The invention provides a DNA adaptor that comprises an RNA polymerase promoter region selected from SP6, T7, or T3. The cytosines of the RNA polymerase promoter are bisulfite-protected, so that promoter cytosines are protected from bisulfite conversion. The adaptor is configured to be used in a bisulfite workflow in which promoter information and sequencing-ready templates are preserved.
The invention further provides a composition that includes the DNA adaptor together with a primer complementary to the RNA polymerase promoter. The promoter region remains SP6, T7, or T3, and the cytosines of the RNA polymerase promoter are bisulfite-protected. The disclosed compositions also include primer-associated and adaptor structural features as part of enabling downstream nucleic-acid processing that supports detection of bisulfite-treated cytosine states.
The invention is described as supporting LABS-seq for whole-genome, unbiased amplification of bisulfite-treated DNA using RNA polymerase promoter adaptors with bisulfite-protected promoter cytosines. In the described workflow, bisulfite-treated DNA is processed with primer hybridization and primer extension to generate double-stranded DNA, followed by in vitro transcription with RNA polymerase for linear amplification, and then reverse transcription and sequencing. The disclosed approach enables detection of 5mC/5hmC and related cytosine modifications by calling 5mC as “C,” and it enables downstream methylation-based profiling.
Claims Coverage
The provided independent claims cover two inventive features: a bisulfite-protected RNA polymerase promoter DNA adaptor, and a composition that includes that adaptor with a primer complementary to the promoter.
Bisulfite-protected RNA polymerase promoter adaptor
A DNA adaptor comprising an RNA polymerase promoter, wherein the cytosines of the RNA polymerase promoter are bisulfite-protected, wherein the RNA polymerase promoter is a SP6, T7, or T3 promoter region.
Promoter-complementary primer adaptor composition
A composition comprising a DNA adaptor comprising a RNA polymerase promoter, wherein the cytosines of the RNA polymerase promoter are bisulfite-protected, further comprising a primer complementary to the RNA polymerase promoter, wherein the RNA polymerase promoter is a SP6, T7, or T3 promoter.
The independent claims focus on the structure of a DNA adaptor with an SP6, T7, or T3 RNA polymerase promoter whose promoter cytosines are bisulfite-protected, and on a composition that includes that adaptor plus a primer complementary to the promoter.
Stated Advantages
Enables detection of 5mC/5hmC and related cytosine modifications by calling 5mC as “C.”
Supports whole-genome, unbiased amplification of bisulfite-treated DNA.
Provides comparative performance outcomes including mapping efficiency, duplication level, and coverage uniformity (as described in the provided content).
Documented Applications
Cancer marker discovery via differential methylation regions (DMRs) using methylation profiles derived from the described approach.
Tissue-of-origin deconvolution using cfDNA methylation profiles based on tissue-specific 5mC DMR profile classification.
Classifying the tissue-of-origin of cfDNA by sequencing and calling 5mC as “C,” followed by tissue-specific 5mC DMR profile classification.
Treating a patient by performing the claimed workflow, classifying the 5mC profile from sequenced DNA, and treating the patient according to that 5mC profile.
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