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Hemi-M methylation-modified primer and application thereof

A methylation and hydroxymethylation technology, applied in the field of molecular biology, to achieve broad application prospects, reduce complex processes, and accurately analyze methylation sites.

Active Publication Date: 2018-11-06
SHENZHEN HAPLOX BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, in the current research, the methylation site can only be known by comparing the sequence to the genome
There is no method that can be used as a control for methylation site mapping analysis

Method used

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  • Hemi-M methylation-modified primer and application thereof
  • Hemi-M methylation-modified primer and application thereof
  • Hemi-M methylation-modified primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] (1) A methylation modification primer, the primer sequence is shown in SEQ ID NO.1:

[0054] Said SEQ ID NO.1 is as follows:

[0055] Primer 1: 5'GA / mdC / TGGAGTT / mdC / AGA / mdC / GTGTG 3'

[0056] The primer modification method is as follows:

[0057] In the above methylated primer sequences, all cytosine (C) was modified to 5-methylcytosine (mdC) during synthesis.

[0058] (2) Sample processing:

[0059] If the sample to be processed is gDNA, use the fragmentation enzyme of NEB Company to fragment 2 μg of sample gDNA, and select 100-250 bp DNA fragments by magnetic beads or gel cutting method. Fragmentase treatment conditions: 37°C, 43min, The DNA after fragment selection was dissolved in 30 μL deionized water;

[0060] If the processed sample is cfDNA, it can be directly used in the subsequent experimental process.

[0061] (3) Use the kit NEB library preparation kit for DNA end repair, add A to the 3' end, and ligate with methylated adapters.

[0062] (4) Cytosine (C...

Embodiment 2

[0079] (1) A methylation tag, the nucleotide sequences of the positive and negative strands of the methylation tag are shown in SEQ ID NO.2 and SEQ ID NO.3:

[0080] SEQ ID NO.2 is Barcode F: 5'NNNNNNNNTGAAT 3'

[0081] SEQ ID NO.3 is Barcode R: 5' -TT / mdC / ANNNNNNNNN 3’

[0082] Among them, Barcode F is reverse complementary to Barcode R, and the phosphate modification at the 5' end of Barcode R When the two strands are annealed, Barcode F hangs thymine at the 3' end and does not undergo phosphorylation at the 5' end.

[0083] Anneal Barcode F and Barcode R to label Barcode 1. Barcode 1 is a double-stranded Oligo. The sequence of each strand is the same as SEQ ID NO.2 and NO.3, except that the two strands are complementary to form a double-stranded structure.

[0084] (2) A methylation modification primer, the primer sequence is as follows:

[0085] SEQ ID NO.1 is Primer 1: 5'GA / mdC / TGGAGTT / mdC / AGA / mdC / GTGTG 3'

[0086] The primer modification method is as follows:

[...

Embodiment 3

[0113] (1) A tag, the nucleotide sequence of which is shown in SEQ ID NO.4:

[0114] SEQ ID NO.4 is Oligo: 5' -CATAG / dU / NNNNNNNNCTATGT 3’

[0115] The 5' end of Oligo is phosphorylated, and the five bases at the 5' end are reverse complementary to the 3' end. After annealing, a stem-loop structure is formed, and the 3' end overhangs the thymine label Barcode 2, as shown in Barcode 2 The nucleotide sequence sequence is SEQ ID NO.4, only self-complementary to form a stem-loop structure after annealing:

[0116] (2) A methylation modification primer, the primer sequence is as follows:

[0117] SEQ ID NO.1 is Primer 1: 5'GA / mdC / TGGAGTT / mdC / AGA / mdC / GTGTG 3'

[0118] The primer modification method is as follows:

[0119] In the above methylated primer sequences, all cytosine (C) was modified to 5-methylcytosine (mdC) during synthesis.

[0120] (3) Sample processing:

[0121] If the sample to be processed is gDNA, use the fragmentation enzyme of NEB Company to fragment 2 μg of s...

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Abstract

The invention provides hemi-M methylation-modified primer and application thereof. The hemi-M methylation-modified primer includes at least one methylation-modified cytosine, wherein methylation modification includes methylation and / or hydroxymethylation modification. The specific methylation-modified primer is designed herein so that combining with dNTP (deoxynucleotide) having dCTP (deoxycytidine triphosphate) replaced with mdCTP (methyl deoxyribocytidine triphosphate) is ingeniously achieved; cytosine protection is performed on sample DNA; the sample DNA is creatively applied to the construction of a methylation library; under the assistance of a specific tag, the sample DNA is applicable to various application fields and scenes; the problem with comparative analysis of library DNA is solved; more accurate methylation sites is achieved. The hemi-M methylation-modified primer and application thereof have a promising application prospect and great market value.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a Hemi-M methylation modification primer and its application. Background technique [0002] In epigenetics, methylation is relatively profound in current research. The methylation level of DNA can determine the expression or inhibition of one or a type of gene, which plays an important role in the normal function of cells, and then affects the life process of human beings. Therefore, methylation research is one of the hot areas of epigenetics research. The methylation at the genomic DNA level is under the action of DNA methyltransferase, using thioadenosylmethionine as a methyl donor, adding a methyl group to the 5'-carbon atom of cytosine to form a 5- Methylcytosine, the principle is as follows figure 1 shown. [0003] CpG island (CpG island) is some regions rich in CpG dinucleotides, with a length of 300-3000bp, CpG is the abbreviation of cytosine (C)-phosphate (p...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6886C40B50/06
CPCC12N15/11C12Q1/6886C12Q2600/154C40B50/06
Inventor 张晓妮屈宏越许明炎陈实富
Owner SHENZHEN HAPLOX BIOTECH
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