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Hydrosulfite-free whole genome methylation analysis

A technology of hydroxymethylcytosine and methylcytosine, which is applied in the field of bisulfite-free genome-wide methylation analysis, can solve the problem that methylation pictures cannot be generated

Pending Publication Date: 2022-03-11
LUDWIG INST FOR CANCER RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it only covers a small subset of CpG sites in the context of a specific sequence and thus does not yield a comprehensive picture of methylation

Method used

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  • Hydrosulfite-free whole genome methylation analysis
  • Hydrosulfite-free whole genome methylation analysis
  • Hydrosulfite-free whole genome methylation analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0247] Example 1: TAPS and WGTAPS

[0248] method

[0249] Preparation of model DNA.

[0250] DNA oligonucleotides for MALDI and HPLC-MS / MS testing. DNA oligonucleotides ("oligonucleotides") with C, 5mC and 5hmC were purchased from Integrated DNA Technologies (IDT). All sequences and modifications can be found at Figure 6 and Figure 7 middle. DNA oligonucleotides with 5fC were synthesized by the C-tailing method: DNA oligonucleotides 5′-GTCGACCGGATC-3′ were annealed to 5′-TTGGATCCGGTCGACTT-3′ followed by 5-formyl-2′-dCTP (Trilink Biotech) and Klenow fragment 3'→5'exo-(New England Biolabs) were incubated in NEB buffer 2 at 37°C for 2 hours. The product was purified with a Bio-Spin P-6 gel column (Bio-Rad).

[0251] DNA oligonucleotides with 5caC were synthesized using the Expedite 8900 DNA synthesis system using the standard phosphoramidite (Sigma) 5-carboxy-dC-CE phosphoramidite (Glen Research). Subsequent deprotection and purification were performed using Glen-Pak...

Embodiment 2

[0385] Example 2: Endonuclease Enriched TAPS (eeTAPS)

[0386] method

[0387] Preparation of Spike Controls.

[0388] 4 kb were prepared by PCR amplification of the pNIC28-Bsa4 plasmid (Addgene, Cat #26103) in reactions containing 1 ng DNA template, 0.5 μM primers, and 1X Phusion High-Fidelity PCR Master Mix (Thermo Scientific) with HF buffer Spike controls. Primer sequences are listed in Table 10. PCR products were purified by Zymo-IC columns (Zymo Research) and methylated with HpaII methyltransferase (New England Biolabs) in 50 μL reactions at 37° C. for 2 hours. Methylated products were purified with 1X Ampure XP beads (Beckman Coulter) according to the manufacturer's protocol. By using M . SssI enzyme (New England Biolabs) methylated unmethylated λ-DNA (Promega) to prepare fully CpG methylated λ-DNA.

[0389] Preparation of vector DNA.

[0390] Vectors were prepared by PCR amplification of the pNIC28-Bsa4 plasmid (Addgene, catalog #26103) in reactions containing ...

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Abstract

The present disclosure provides a method for the cost effective bisulfite-free identification of the position of one or more of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxycytosine, and 5-formylcytosine in DNA, including whole genome DNA, and a method for the cost effective bisulfite-free identification of the position of one or more of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxycytosine, and 5-formylcytosine. The methods described herein are based on the conversion of a modified cytosine (5mC, 5hmC, 5fC, 5caC) to a dihydrouracil (DHU), e.g. By a TET-assisted pyridine borane treatment, followed by cleavage of the DHU with an endonuclease and identification of a cleavage site corresponding to the position of the modified cytosine.

Description

technical field [0001] The present disclosure provides methods for genome-wide identification of the location of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxycytosine, and / or 5-formylcytosine. Background technique [0002] 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are two major epigenetic markers present in mammalian genomes. 5hmC is generated from 5mC by the ten-eleven translocation (TET) family of dioxygenases. Tet can further oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), which are much less abundant in mammalian genomes than 5mC and 5hmC (than the abundance of 5hmC 10 to 100 times lower). Together, 5mC and 5hmC play critical roles in a wide range of biological processes, from gene regulation to normal development. Aberrant DNA methylation and hydroxymethylation are associated with various diseases and are well-established hallmarks of cancer. Therefore, the determination of 5mC and 5hmC in DNA sequences is not only importa...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/6827
CPCC12Q1/6869C12Q1/6827C12Q2521/531C12Q2523/115C12Q2521/301C12Q2525/117C12Q2525/191C12Q2600/154C12Q1/6806C12Q1/683
Inventor 宋春啸程京飞波琳娜·西耶卡-杰琳斯卡刘艺斌
Owner LUDWIG INST FOR CANCER RES
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