METHOD FOR GENOMIC PROFILING OF DNA 5-METHYLCYTOSINE AND 5-HYDROXYMETHYLCYTOSINE

• US 20170253924A1
• Filed: 04/26/2017
• Published: 09/07/2017
• Est. Priority Date: 03/02/2016
• Status: Active Grant

• Current Assignee: Beijing Xuanniao Feixun Technology Company Limited
• Original Assignee: Shanghai Epican Genetech Co., Ltd.
• Inventors: Lu, Xingyu, Song, Yanqun
• Granted Patent: US 11,162,139 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

The present invention provides a method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine, comprising the following steps: (1) DNA purification and fragmentation pretreatment: the target DNA is extracted and then broken to an average of 50 nucleotides to 10,000 nucleotides in length; (2) the repair of trace amount of DNA and the ligation thereof to the adaptor: the pre-treated DNA fragments are repaired and ligated with the sequencing adaptor required for the second-generation sequencing, (3) covalently labeling 5-methylcytosine and 5-hydroxymethylcytosine, (4) solid-phase enrichment of the labeled DNA fragments having cytosine with 5-position modification; (5) the PCR amplification of the solid-phase enriched DNA fragments, the PCR product is obtained and purified to obtain a library for the second-generation sequencing, after mapping the sequencing reads to the genome, the distribution map of the cytosine with 5-position modification in the DNA sample could be generated. The present invention greatly enhances the selectivity and efficiency of binding of the solid-phase surface with the DNA modified base.

Claims

1. A method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine, characterized in that, it comprises the following steps:

(1) DNA purification and fragmentation pretreatment;

the target DNA is extracted using a mechanical force or digestive enzyme to break it to an average of 50 nucleotides to 10,000 nucleotides in length;

(2) the repair of trace amount of DNA and the ligation thereof to the adaptor;

the pre-treated DNA fragments are repaired and ligated with the sequencing adaptor required for the second-generation sequencing;

(3) covalently labeling 5-methylcytosine and 5-hydroxymethylcytosine;

cytosine with 5-position modification in the DNA fragment to which the adaptor is ligated is covalently labeled;

(4) solid-phase enrichment of the labeled DNA fragment having cytosine with 5-position modification;

the labeled DNA fragment having cytosine with 5-position modification is bound to the solid phase in the binding buffer, and the solid phase surface is repeatedly washed, to remove the unbound DNA fragment;

(5) the PCR amplification primers corresponding to the adaptor are used to carry out an amplification of the solid-phase enriched DNA fragments, the PCR product is obtained and purified to obtain a library for the second-generation sequencing, after mapping the sequencing reads to the genome, the distribution map of the cytosine with 5-position modification in the DNA sample could be generated.

2. The method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine according to claim 1, characterized in that,

wherein, in step (1), the DNA fragment is derived from the free DNA fragments in a body fluid, or genomic DNA purified from tissues, cells and organelles.

3. The method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine according to claim 2, characterized in that,

in step (1) the body fluid of DNA fragment is derived from blood, urine, sweat, sputum, feces, cerebrospinal fluid, ascites, pleural effusion, bile, pancreatic fluid.

4. The method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethyleytosine according to claim 1, characterized in that,

wherein, the DNA fragment repair in the step (2) is a step of repairing base damage in DNA fragment, and 5’

and 3′

of DNA are made up to a blunt-end.

5. The method for genornic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine according to claim 1, characterized in that,

in step (3), the method of labeling, comprises the steps of:

i) an azide-group modified sugar is covalently linked to the hydroxymethl group of 5-hydroxymethylcytosine using transglycosidase, ii) azido sugar-modified 5-hydroxymethylcytosine reacts with click-chemistry substrate linked directly or indirectly to a biotin.

6. The method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine according to claim 5, characterized in that

the Transglycosylase includes, but is not limited to, bGT, aGT and its derivatives, analogs or recombinant enzymes;

azide-group modified sugars include, but are not limited to, 6-N3-glucose or other azido-modified sugar.

7. The method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine according to claim 5, characterized in that,

labeling methylcytosine includes the steps of i) 5-methylcytosine oxidized to 5-hydroxymethylcytosine using murine Tet oxidase or a derivative thereof, analogues, recombinant enzymes, biotin labeling of 5-hydroxymethylcytosine is carried out as described above.

8. The method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine according to claim 1, characterized in that,

wherein, labeling steps of 5-hydroxymethylcytosine can be carried out in sequence or simultaneously in a reaction.

9. The method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine according to claim 1, characterized in that,

wherein, in step (4), the solid phase material includes a magnetic ball having a diameter of 1 nm to 100 μ

m, agarose beads of 1 nm to 100 μ

m in diameter, artificial macromolecule balls with diameters of 1 nm to 100 μ

m, silicon wafers with surface modification or other biochips.

10. The sequencing method of DNA 5-methylcytosine and 5-hydroxy methylcytosine according to claim 1, characterized in that,

in step (5), the qualified sequencing library is obtained by amplification of 1-40 PCR cycles on the solid phase directly, or after the amplification by 1-40 PCR cycles the solid-liquid phase is separated and then the qualified sequencing library is obtained by further amplification of 1 to 40 PCR cycles.