Methods for the amplification of bisulfite-treated DNA
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.
1. A method for amplifying a deoxyribonucleic acid (DNA) molecule comprising:
(a) ligating an adaptor to the DNA molecule to generate a ligated DNA molecule, wherein the adaptor comprises a RNA polymerase promoter region comprising bisulfite-protected cytosines;
(b) treating the ligated DNA molecule with bisulfite to generate a bisulfite-treated DNA molecule;
(c) hybridizing a primer to the bisulfite-treated DNA molecule;
(d) extending the primer to make a double stranded DNA molecule; and
(e) in vitro transcribing the double-stranded DNA molecule to make a RNA molecule.
2. The method of claim 1 , wherein the bisulfite-protected cytosines comprise 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC).
3. The method of claim 1 , wherein the bisulfite-protected cytosine comprises a oxime or hydrazone-modified 5-formylcytosine (5fC).
4. The method of claim 1 , wherein the bisulfite-protected cytosines comprise a bisulfate-protected 5-carboxylcytosine (5caC).
5. The method of claim 4 , wherein the 5caC is an amide-modified 5caC.
6. The method of claim 1 , wherein the RNA polymerase promoter region is a SP6, T7, or T3 promoter region.
7. The method claim 1 , wherein (a) comprises end modification of the DNA and/or end repair of the DNA.
8. The method of claim 1 , wherein the adaptor further comprises a 3′ end-blocked molecule.
9. The method of claim 1 , wherein the adaptor is partially double-stranded.
10. The method of claim 1 further comprising one or more purification steps.
11. The method of claim 1 further comprising reverse transcription of the RNA molecule of (e) to make a corresponding DNA molecule.
12. The method of claim 11 further comprising sequencing the corresponding DNA molecule.
13. A method for evaluating genomic DNA from a single cell comprising performing the method of claim 12 , wherein the DNA molecule is from the genomic DNA of the single cell.
14. The method of claim 1 , wherein the DNA molecule is 100-300 bp in length.
15. The method of claim 1 , wherein the method excludes a DNA enrichment step.
16. The method of claim 1 , wherein the DNA molecule is a cell-free DNA (cfDNA) molecule.
17. The method of claim 1 , wherein the DNA molecule is a genomic DNA molecule.
18. The method of claim 1 , wherein the DNA molecule was isolated from a biopsy sample.
19. The method of claim 18 , wherein the biopsy sample is a liquid sample.
20. The method of claim 1 , wherein the RNA polymerase promoter region is a T7 promoter region.
21. The method of claim 1 , further comprising, prior to (a), (1) modifying a 5-hydroxymethylcytosine (5hmC) in the DNA molecule to protect it from oxidation; and (2) oxidizing the DNA molecule with a methylcytosine dioxygenase.
22. The method of claim 1 , further comprising, prior to (a), oxidizing the DNA molecule with an oxidant.