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Preparation method of molecular tag

A molecular tag and sequence technology, applied in the field of DNA variation detection, can solve the problems of data unavailability, long operation cycle, and lower data efficiency, and achieve the effects of improving reaction rate, evaluating preference, and reducing waste

Inactive Publication Date: 2016-11-16
首度生物科技(苏州)有限公司 +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Double sequencing technology uses 12bp random primers and 4bp or 5bp fixed sequences, which leads to the unavailability of the first 16-17bp data in DNA sequencing data, reducing the data efficiency; CAGT fixed sequences are not helpful for distinguishing DNA molecular sequences, resulting in The quality is reduced; this method is limited to all αβ family and βα family reads, and the mutation sites that exist in the double-stranded DNA fragments are the real mutation sites; and the improved method of double sequencing technology, through the internal enzyme digestion method in the The T base is introduced at the 3′ end, the reaction time is 16 hours, the operation cycle is long, and the efficiency of the enzyme digestion reaction is low

Method used

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  • Preparation method of molecular tag

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Embodiment 1

[0037] 1. Synthesis and annealing of the adapter

[0038](1) Take a tube of sequencing adapter DNA whose sequences are 5'-TACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' and 5'-TCTTTCTACAGTCANNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3', where NNNNNN in the sequence is a 6bp random barcode, where N means A / T / G / Any base in C will do. That is, the sequences contained in the adapter DNA are at most 4 to the 6th power, and the difference of these sequences depends on what base N is. Centrifuge at 20,000 g for 10 min at 4°C to ensure that the adapter powder gathers at the bottom of the tube, and the labels are adapter-1 and adapter-2 respectively.

[0039] (2) Open the cap of the tube. At this time, be careful not to let the adapter powder float out of the tube. Add Nuclease-Free water (nuclease-free water) to adapter-1 and adapter-2 to dissolve the powder to make the concentration 100nmol / mL, that is, 100μM; then store at -20°C, if not used for a long time, store at -80°C Save it.

[004...

Embodiment 2

[0073] 1. Synthesis and annealing of the adapter

[0074] (1) Take a tube of sequencing adapter DNA whose sequences are 5'-TACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' and 5'-TCTTTCTACAGTCANNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3', where NN in the sequence is a 2bp random barcode, where N represents A / T / G / Any base in C will do. Centrifuge at 20,000 g for 10 min at 4°C to ensure that the adapter powder gathers at the bottom of the tube, and the labels are adapter-1 and adapter-2 respectively.

[0075] (2) Open the cap of the tube. At this time, be careful not to let the adapter powder float out of the tube. Add Nuclease-Free water (nuclease-free water) to adapter-1 and adapter-2 to dissolve the powder to make the concentration 100nmol / mL, that is, 100μM; then store at -20°C, if not used for a long time, store at -80°C Save it.

[0076] (3) Take 25 μL of adapter-1 (100 μM) and 25 μL of adapter-2 (100 μM) and mix them uniformly to obtain 50 μL of a mother solution with a concentration...

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Abstract

The invention relates to a preparation method of a molecular tag. The preparation method comprises the following steps: introducing a barcode sequence to a 5' adaptor terminal of a DNA molecular template; extending a 3' adaptor terminal by virtue of DNA polymerase, and complementing the 3' adaptor terminal with the barcode sequence, so that a complementary sequence is obtained; adding DNA polymerase to the complementary sequence, and introducing thymine to a 3' adaptor terminal of the complementary sequence, so that a modified adaptor is obtained; breaking to-be-detected DNA into DNA short segments, and linking the DNA short segments to the modified adaptor by virtue of DNA ligase, so that a DNA ligation product is obtained; amplifying the DNA ligation product by virtue of a polymerase chain reaction, so that an amplification product is obtained; and sequencing the amplification product, then contrasting the amplification product to a human reference genome, and selecting a DNA sequence, which has the minimum base error rate, from a plurality of DNA template copies, so that the molecular tag is obtained. The molecular tag technology, by changing a barcode length, can remove fixed sequences, reduce data waste in sequencing and accelerate a reaction rate, and wrong and actual DNA variations in a DNA amplification process can be effectively distinguished.

Description

technical field [0001] The invention relates to the field of DNA variation detection, in particular to a method for preparing a molecular label. Background technique [0002] Compared with traditional detection methods, liquid biopsy technology is a hot technology in the field of tumor precision medicine because of its advantages of less side effects, simple operation, and repeatable sampling. Its main detection objects include: circulating tumor cells (CTCs), circulating Tumor DNA (ctDNA) and tumor exosomes can prompt information such as tumor development and drug resistance, and guide individualized drug treatment. [0003] ctDNA is a kind of free extracellular single-stranded or double-stranded DNA existing in plasma, serum, cerebrospinal fluid and other body fluids, mainly from necrotic or apoptotic tumor cells, exosomes secreted by tumor cells, and circulating tumor cells. The size of ctDNA fragments is usually 160-180bp, with a half-life of 2 hours in the blood, carry...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王海龙胡妮徐健唐元华
Owner 首度生物科技(苏州)有限公司
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