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A method for extracting cfDNA from follicular fluid

A technology of follicular fluid and lysate, which is applied in the field of genetic testing, can solve the problems of many fragments, less research, and complex components of follicular fluid, and achieve the effects of saving time and cost, simplifying extraction steps, and reducing waste

Active Publication Date: 2022-07-15
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of high-throughput sequencing is based on the construction of high-quality gene libraries. However, due to the complex components in follicular fluid, the existing kits based on silica gel membrane columns are more suitable for the extraction of cfDNA from serum and pleural fluid, but cannot be extracted from follicular fluid. Obtain high-quality cfDNA, the quality inspection results show that there are many and complex fragments, no obvious main peaks, and it is impossible to carry out subsequent experimental operations such as the construction of subsequent sequencing libraries, such as figure 1 As shown, there are few studies on follicular fluid cfDNA based on high-throughput sequencing technology, which is still in its infancy

Method used

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  • A method for extracting cfDNA from follicular fluid
  • A method for extracting cfDNA from follicular fluid
  • A method for extracting cfDNA from follicular fluid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Extraction of cfDNA

[0039] 1. Separation and preservation of follicular fluid

[0040]The centrifuge was pre-cooled at 4°C, centrifuged at 3000g for 15min, and the supernatant was carefully aspirated. The supernatant was then centrifuged at 16,000g for 10 min, and the supernatant was transferred to a new centrifuge tube. It can be stored at 4°C if used on the same day, or at -80°C if stored for a long time.

[0041] 2.1, configure the lysate

[0042] 100 mM Tris-HCl, 20-30 mM EDTA, 500 mM NaCl, 1% SDS, along with 6 μg Carrier RNA per ml, vortex to mix.

[0043] 2.2, cracking

[0044] Take a new 15ml centrifuge tube, add 100μl proteinase K (1mg / ml), then add 1ml follicular fluid and 800μl lysate in sequence, vortex at maximum speed for 30s, and incubate the centrifuge tube at 60°C for 30min.

[0045] 3. Adsorption of cfDNA by carboxyl magnetic beads

[0046] Add 0.5ml of isopropanol to the above centrifuge tube, after vortexing for 30s, add 200μl carbox...

Embodiment 2

[0060] Example 2 Construction of high-throughput sequencing library

[0061] (1) cfDNA end repair

[0062] Add the corresponding samples and reagents to the PCR tube according to the reaction system in Table 1, vortex to mix well, and then put them in the PCR machine, and incubate at 20 °C for 5 min. After the reaction is over, add ddH 2 O to the final volume of the system 200 μl. Then transfer it to a new 1.5ml centrifuge tube, add 200μl of mixed phenol (phenol:chloroform:isoamyl alcohol=25:24:1, pH=8.0) to the system (the same volume as the sample to be purified), shake vigorously Mix well and centrifuge at 13000rpm for 5min at room temperature. Transfer the supernatant to a new 1.5ml tube (be careful not to aspirate the protein layer at the junction of the aqueous phase and the organic phase at this time to avoid contamination of genomic DNA by aspirated protein. It is not necessary to completely absorb the aqueous phase, and leave a little to avoid contamination). Add ...

Embodiment 3

[0085] Example 3 High-throughput sequencing

[0086] High-throughput sequencing of the high-throughput sequencing library of cfDNA in Example 2 was performed using Illumina Hiseq 2000. The sequencing data was used for basic quality inspection using Fastqc software, and the data quality was qualified before further use. The PCR primer sequences used for sequencing are shown in Table 6.

[0087] Table 6 PCR primer sequences

[0088]

[0089] The quality inspection results of paired-end sequencing are as follows: Figure 5 As shown, most of the base quality is above 30, indicating that the data quality is good. The meaning of the bar is 25%-75% of the data distribution, and only the data whose total quality score is above 30 (representing a sequencing error rate of 1 in 1,000) can be further analyzed.

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Abstract

The invention discloses a method for extracting cfDNA from follicular fluid. The follicular fluid sample is centrifuged in two steps to remove excess cell debris; the follicular fluid is lysed with a lysing solution, the added proteinase K digests the excess protein, and at the same time the added Carrier RNA makes DNA more likely to precipitate and settle; use carboxyl magnetic beads to adsorb DNA; under conditions of high salt and low pH, cfDNA in follicular fluid is adsorbed to magnetic beads, and after salt washing and alcohol washing, the cfDNA below 100 bp is removed After elution, 0.5 times the volume of carboxyl magnetic beads was added again to adsorb and remove long fragments greater than 300 bp to obtain cfDNA with a suitable length range. At the same time, end-repair the obtained cfDNA to generate blunt-end ligation, add base A at the 3' end, and add adapters at both ends of the cfDNA, amplify and purify, etc., to complete the construction of a high-throughput library, and finally complete the machine. Sequencing.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a method for extracting cfDNA from follicular fluid, a kit and a constructed cfDNA library. Background technique [0002] The occurrence of follicles is an extremely complex and delicate process, which involves not only the interaction and connection between various cells, but also the changes and influences of the biochemical components of local follicular fluid. The follicular fluid, as the microenvironment for the survival of oocytes, directly affects the growth and development process, fertilization ability and developmental potential of oocytes. Many studies regard it as an auxiliary indicator for evaluating follicle development ability, which is of great significance for further screening of high-quality embryos and the successful establishment of clinical pregnancy. Follicular fluid has attracted much attention because it is easy to obtain and contains many factors related t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 康亚妮秦玉兰毛湛睿赵晖
Owner SHANGHAI JIAOTONG UNIV
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