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Methylation detection composition, kit and method

A detection method and sequence-combining technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of reducing sequence complexity, difficult primer design, labor-intensive and time-consuming, etc.

Pending Publication Date: 2022-02-22
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are many deficiencies in the method based on bisulfite conversion: 1. DNA samples converted by bisulfite need to undergo harsh chemical treatment processes, such as low pH, high temperature, high concentration of bisulfite, etc., these conditions will lead to A large amount of DNA is degraded, the degradation is as high as 90%, and the integrity of the template is reduced, so the required initial amount of cfDNA is large; 2. Incomplete bisulfite treatment will lead to incomplete conversion of unmethylated cytosine and false positives 3. Unmethylated cytosine accounts for about 95% of the total cytosine in the human genome, and converting all unmethylated cytosine to uracil will It will seriously reduce the complexity of the sequence. When simultaneously detecting multiple target molecules, the design of primers is more difficult, which may lead to a decrease in detection accuracy and increase detection costs; 4. The bisulfite conversion process takes a long time and requires repeated washing Purification and cumbersome operation consume a lot of manpower and time

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  • Methylation detection composition, kit and method
  • Methylation detection composition, kit and method
  • Methylation detection composition, kit and method

Examples

Experimental program
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Effect test

specific Embodiment

[0142] Specific examples The methylation site in the sequence described in SEQ ID NO: 10 of the Septine 9 gene is detected by the following method:

[0143]Use the oligonucleotide linker (1) shown in SEQ ID NO:1 or 6 to contact with the cfDNA digested by GlaI, the oligonucleotide linker (1) and the target molecule (2) shown in SEQ ID NO:10 Complementary binding; the target molecule (2) is extended using SEQ ID NO: 1 or 6 as a template, and a sequence complementary to SEQ ID NO: 1 or 6 is added to the 3' end of the target molecule (2) to obtain an extended target molecule (3 ); the extended target molecule (3) is complementary to the capture oligonucleotide (4) shown in SEQ ID NO: 2 or 7; the capture oligonucleotide (4) is carried out with the extended target molecule (3) as a template An extension reaction, adding a sequence complementary to the extended target molecule (3) at the 3' end of the capture oligonucleotide (4) to obtain an extended capture oligonucleotide (5); the ...

Embodiment 1

[0152] Example 1: Methylation detection of ultrasonically fragmented DNA of human Septine 9 gene

[0153] (1) Genomic DNA of Jurkat cell line and Hela cell line were extracted respectively, and the methylation status of Septine9 gene was identified by sequencing.

[0154] (2) Ultrasonic treatment of the above two genomic DNAs to obtain fragmented DNA with a fragment size of about 150 bp.

[0155] (3) Use the methylation-dependent restriction endonuclease GlaI to carry out enzyme digestion reaction on the above-mentioned ultrasonically fragmented DNA. The reaction system is: 1× enzyme digestion buffer, 10U GlaI, ultrasonically fragmented DNA at different concentrations, and the total volume is 10 μL; the reaction condition is to incubate at 37°C for 1 hour; after the enzyme digestion reaction, heat the system to 85°C and incubate for 10 minutes to heat inactivate GlaI.

[0156] (4) Add the Septine 9 gene oligonucleotide linker and capture oligonucleotide to the above enzyme di...

Embodiment 2

[0175] Embodiment 2: Experiment of different structural oligonucleotide linkers

[0176] In order to reduce the competition between oligonucleotide adapters, capture oligonucleotides and target molecules, in this example, oligonucleotide adapters with different structures were selected for detection. The capture oligonucleotides, specific primers, universal primers and specific probes used are the same as those in Example 1, and the restriction conditions and amplification conditions are the same as those in Example 1.

[0177] Oligonucleotide adapters used include:

[0178] Linear Oligonucleotide Adapter

[0179] 5-TGCCGTCAGAGTCCTGTCTCGAGCGACCCGCTGCCCACCAG (SEQ ID NO: 1)

[0180] Self-folding oligonucleotide adapters (underlined are self-folding regions)

[0181] 5- TCGAGACAGGAC TGCCGTCAGA GTCCTGTCTCGA GCGACCCGCTGCCCAC (SEQ ID NO: 6)

[0182] The partial sequence of the human Septine 9 gene is shown in Example 1.

[0183] Such as image 3As shown, using straight-cha...

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Abstract

The invention relates to a methylation detection composition, kit and method, and specifically provides oligonucleotide linkers and a capture oligonucleotide for nucleic acid amplification, use method and use thereof. The oligonucleotide linkers comprise a first binding sequence and a second binding sequence, wherein the first binding sequence comprises a sequence which is the same as a 3' terminal sequence of the capture oligonucleotide, the second binding sequence is complementary with a target molecule, and the capture oligonucleotide comprises a first universal sequence, a folding sequence and a binding capture sequene. According to the invention, the product and the method can realize DNA methylation detection with good specificity and high sensitivity.

Description

technical field [0001] The invention relates to a methylation detection composition, kit and method. Background technique [0002] Plasma contains cell-free DNA (cfDNA). Plasma samples can be taken multiple times and are non-invasive compared to tissue biopsies. However, the content of cfDNA in body fluids is very small, and the content of cfDNA in healthy human plasma is only about 5-10ng / ml. Therefore, the biggest technical difficulty of cfDNA methylation detection lies in how to use a small amount of cfDNA samples to detect a very small amount of abnormal methylation genes. [0003] Currently, methods based on bisulfite conversion are the mainstream choice for cfDNA methylation detection. The principle is that when DNA is treated with bisulfite, cytosine will be deaminated and transformed into uracil, and 5-methylcytosine will keep the original sequence unchanged because the methyl group it carries inhibits the action of bisulfite. The difference in the modification g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2523/301C12Q2521/331C12Q2525/191C12Q2525/186C12Q2563/107Y02A50/30
Inventor 徐高连丘佳妮杨浩徐宏古宏晨
Owner SHANGHAI JIAO TONG UNIV
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