IP Library › Granted Patent US 12,606,866
Granted Patent B2
US 12,606,866 · App. 18/410,490 · Granted Apr 21, 2026

Methods for the amplification of bisulfite-treated DNA

Inventors: Chuan He (Chicago, IL); Ji Nie (Chicago, IL); Xiao-Long Cui (Chicago, IL)
Assignee: THE UNIVERSITY OF CHICAGO
C12Q1/6865
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Quick Facts
Patent No.
US 12,606,866
App. No.
18/410,490
Granted
Apr 21, 2026
Kind
B2
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.

Claims (35)

1 . A DNA adaptor comprising a 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.

2 . The DNA adaptor of claim 1 , wherein the bisulfite-protected cytosines comprise 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC).

3 . The DNA adaptor of claim 1 , wherein the bisulfite-protected cytosines comprise a oxime or a hydrazone-modified 5-formylcytosine (5fC).

4 . The DNA adaptor of claim 1 , wherein the bisulfite-protected cytosines comprise a bisulfate-protected 5-carboxylcytosine (5caC).

5 . The DNA adaptor of claim 1 , wherein the adaptor comprises a 3′ end-blocked molecule.

6 . The DNA adaptor of claim 1 , wherein the adaptor is partially double-stranded.

7 . A composition comprising the DNA adaptor of claim 1 .

8 . The composition of claim 7 , further comprising genomic or cell-free (cfDNA).

9 . The composition of claim 7 , further comprising a ligase.

10 . The composition of claim 7 , further comprising a DNA polymerase or RNA polymerase.

11 . The composition of claim 7 , further comprising a primer complementary to the RNA polymerase promoter.

12 . A method of identifying a tissue-of-origin of cfDNA comprising,

(a) ligating the adaptor of claim 1 to the cfDNA molecule to generate a ligated cfDNA molecule;

(b) treating the ligated cfDNA molecule with bisulfite to generate a bisulfite-treated cfDNA molecule;

(c) hybridizing a primer to the bisulfite-treated cfDNA molecule;

(d) extending the primer to make a double stranded cfDNA molecule;

(e) in vitro transcribing the double-stranded cfDNA molecule to make a RNA molecule;

(f) reverse-transcribing the RNA to make cDNA;

(g) sequencing the cDNA and identifying the 5mC in the sequenced cDNA as a “C” in the sequence;

(h) identifying a tissue-specific 5mC differentially methylated regions (DMRs) profile in the sequenced cDNA; and

(i) classifying the cfDNA tissue-of-origin from the DMR profile.

13 . A method of treating a patient comprising performing the method of claim 12 , and treating the patient according to the tissue-of-origin classification from the DMR profile, wherein the cfDNA molecules are provided from a biological sample from the patient.

14 . A method for identifying 5mC in DNA molecules comprising:

(a) ligating the adaptor of claim 1 to the DNA molecules;

(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;

(f) reverse-transcribing the RNA to make DNA;

(g) sequencing the DNA and identifying the 5mC in the sequenced DNA as a “C” in the sequence.

15 . A method of treating a patient comprising performing the method of claim 14 , classifying the 5mC profile from the sequenced DNA, and treating the patient according to the 5mC profile, wherein the DNA molecules are provided from a biological sample from the patient.

16 . 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.

17 . The composition of claim 16 , wherein the bisulfite-protected cytosines comprise 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), a oxime or a hydrazone-modified 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC).

18 . The composition of claim 16 , wherein the adaptor comprises a 3′ end-blocked molecule.

19 . The composition of claim 16 , further comprising genomic or cell-free (cfDNA), a ligase, a DNA polymerase or RNA polymerase, and/or a primer complementary to the RNA polymerase promoter.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 9, 2024
From: HE, CHUAN; NIE, JI; CUI, XIAO-LONG
To: THE UNIVERSITY OF CHICAGO
Reel/Frame 066425/0897 →
Continuity (4)
Division 18057424 · Nov 21, 2022
Division 17250365
Provisional Application 62711184 · Jul 27, 2018
Related Publication 20240209430A1 · Jun 27, 2024
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