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Reagent for constructing free DNA library and application thereof

A DNA library and reagent technology, applied in the field of reagents for building cell-free DNA libraries, can solve problems such as interference with embryo-derived DNA signals, and achieve the effects of shortening library construction time, reducing library construction costs, and improving detection accuracy

Active Publication Date: 2022-02-11
SUZHOU BASECARE MEDICAL DEVICE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If according to the current niPGT detection method, the blastocyst culture fluid sample is first subjected to single-cell whole-genome amplification, the amount of genes in the sample is increased to meet the needs of library construction, and then library construction and high-throughput sequencing are performed, maternal DNA will be The further amplification of the background signal of the maternal source pollution leads to a very strong background signal, which seriously interferes with the DNA signal from the embryo, and even directly covers the signal of the embryo. For example, CN111440857A discloses a method for noninvasive preimplantation genetic testing The method uses the above-mentioned kit to perform whole-genome amplification on the blastocyst culture fluid sample, conduct short tandem repeat sequence analysis on the amplified product and DNA samples from both parents to detect maternal contamination, and perform library preparation and next-generation sequencing on the amplified product. Tests to determine if there is an abnormal number of chromosomes

Method used

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  • Reagent for constructing free DNA library and application thereof
  • Reagent for constructing free DNA library and application thereof
  • Reagent for constructing free DNA library and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] The present embodiment prepares mouse blastocyst culture liquid sample, comprises the following steps:

[0077] (1) Embryos are fertilized, and fertilized eggs are obtained according to the standard IVF cycle operation procedure of the reproductive center;

[0078] (2) Embryo transfer and single blastocyst culture, transfer the embryos to the marked blastocyst culture dish for single droplet culture (culture medium volume 20 μL);

[0079] (3) Collection of samples of blastocyst culture fluid. Place the embryos after the fluid change in the incubator to continue culturing. When the blastocysts develop to stage 4, evaluate the grade of the embryos, select embryos with 4CB / 4BC or above, and the blastocysts are fully expanded, and collect the corresponding ones. blastocyst culture medium;

[0080] Take 10 tubes of blastocyst culture fluid samples of mouse embryos above stage 4 and mix them to a total of 200 μL, vortex and mix 3 times for 3 seconds each time, and then distr...

Embodiment 2

[0082] This embodiment utilizes the mouse blastocyst culture fluid prepared in Example 1 to construct a library, and the construction method of the library comprises the following steps:

[0083] (1) Sample Lysis

[0084] 1) Centrifuge the 0.2mL PCR tube containing the blastocyst culture solution sample at 2000 rpm for 10 s, place it on ice, prepare the system according to Table 4, vortex for 5 s, mix the solution and centrifuge at 2000 rpm for 10 s, synchronously Set negative control, replace blastocyst culture medium with nuclease-free water; positive control, replace blastocyst culture medium with 8 mouse blastocyst cells;

[0085] Table 4

[0086]

[0087] 2) Set the reaction program of the PCR instrument, and perform lysis as shown in Table 5.

[0088] table 5

[0089]

[0090] (2) End repair plus dA tail

[0091] 1) Add 19 μL of nuclease-free water and 1 μL of carrier DNA (SEQ ID NO.1) to the lysate of step (1);

[0092] 2) Prepare the end repair reaction soluti...

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Abstract

The invention discloses a reagent for constructing a free DNA library and application of the reagent. The reagent comprises carrier DNA, heat-sensitive protease and a linker connecting reagent, wherein the linker connecting reagent comprises ligase, a connecting buffer solution, propylene glycol and polyethylene glycol. By utilizing the reagent, the problem that high-throughput sequencing library construction can be carried out only after single-cell whole genome amplification in the prior art can be solved, and the problems of polluted DNA signal amplification and covering of information such as cfDNA methylation sites, cfDNA length and cfDNA sequence characteristics caused by single-cell whole genome amplification are avoided; the library building time is obviously shortened; and the library building cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a reagent for constructing a free DNA library and an application thereof. Background technique [0002] In 2013, Professor S. Stigliani and his team detected embryo-derived genomic DNA and mitochondrial DNA in discarded blastocyst culture medium for the first time (Stigliani, S., et al., Mitochondrial DNA content inembryo culture medium is significantly associated with human embryofragmentation . Hum Reprod, 2013. 28(10): p. 2652-60.), Shamonki and his research team first used D3-D5 / 6 embryo culture medium for non-invasive preimplantation genetic test (Non-invasive Preimplantation Genetic Test for Embryo Aneuploidy, niPGT-A), initially proved the feasibility of non-invasive preimplantation genetic testing (PGT-A) using cell-free DNA in waste embryo culture medium (Shamonki, M.I., et al., Proof of concept: preimplantation genetic screening without embryobiopsy through ...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6806
CPCC40B50/06C12Q1/6806
Inventor 许瑞霞杨祖郭庆凯王莹莹杨玉妍孔令印梁波
Owner SUZHOU BASECARE MEDICAL DEVICE CO LTD
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