阅读说明：本技术 一种提取血浆游离dna的试剂盒和方法 (Kit and method for extracting free DNA (deoxyribonucleic acid) in blood plasma ) 是由 宋新文 李香玲 冯冰青 耿亮 辛文 于 2021-04-25 设计创作，主要内容包括：本发明公开一种提取血浆游离DNA的试剂盒和方法。本发明首先提供了提取血浆游离DNA的试剂盒,所述试剂盒包括裂解液、结合液、清洗液、洗涤液、洗脱液、磁珠和蛋白酶K。本发明进一步提供了上述试剂盒在提取血浆游离DNA中的应用及利用上述试剂盒提取血浆游离DNA的方法。本发明通过对试剂盒中裂解液、结合液、清洗液、洗涤液和洗脱液的优化,尤其是对裂解液、结合液、清洗液的中多种胍盐的组合以及去污剂与异丙醇浓度配比的优化,使该试剂盒最大程度结合血浆游离DNA,去除杂质,使提取得到的血浆游离DNA中无大片度残留,且具有更高的纯度,在下游文库构建实验中具有更高的文库构建效率,更高的测序数据质量,更高的准确性。(The invention discloses a kit and a method for extracting free DNA of blood plasma. The invention firstly provides a kit for extracting free DNA of blood plasma, which comprises lysis solution, binding solution, cleaning solution, washing solution, eluent, magnetic beads and proteinase K. The invention further provides application of the kit in extracting free DNA of blood plasma and a method for extracting the free DNA of the blood plasma by using the kit. According to the invention, through optimizing the cracking liquid, the combination liquid, the cleaning liquid, the washing liquid and the eluent in the kit, especially optimizing the combination of various guanidine salts in the cracking liquid, the combination liquid and the cleaning liquid and the concentration ratio of the detergent to the isopropanol, the kit is combined with plasma free DNA to the maximum extent, impurities are removed, the extracted plasma free DNA has no large residual fragment and has higher purity, and the kit has higher library construction efficiency, higher sequencing data quality and higher accuracy in a downstream library construction experiment.)

1. A kit for extracting free DNA of blood plasma is characterized by comprising lysis solution, binding solution, cleaning solution, washing solution, eluent, magnetic beads and proteinase K;

the lysis solution contains at least two of 2-6mol/L guanidine hydrochloride, 0.5-1.2mol/L guanidine isothiocyanate and 0.5-2.4mol/L sodium iodide, 10-100mmol/L Tris-acetic acid, 10-100mmol/L EDTA, at least one of 5-15% volume fraction Tween-80 and NP-40, and 400-800mmol/L NaCl;

the binding solution contains at least two of 2-6mol/L guanidine hydrochloride, 0.5-1.6mol/L guanidine isothiocyanate and 0.5-1.5mol/L sodium iodide, 10-100mmol/L Tris-acetic acid, 10-100mmol/L EDTA, 0.05-0.5% of PEG6000 by volume fraction, 10-15% of at least one of Tween-80 and NP-40 by volume fraction, and 30-70% of isopropanol by volume fraction;

the cleaning solution contains at least two of 2-6mol/L guanidine hydrochloride, 0.6mol/L guanidine isothiocyanate and 0.6mol/L sodium iodide, 10-100mmol/L Tris-acetic acid, 10-50mmol/L EDTA, at least one of 8-10% volume fraction Tween-80 and NP-40, and 30-50% volume fraction isopropanol;

the washing solution contains 5-20mmol/L Tris-acetic acid, 0.01-0.05% volume fraction of Triton X-100 or Tween-80 and 80% volume fraction of absolute ethyl alcohol;

the eluent contains 5-10mmol/L Tris-acetic acid, 0.05-0.3mmol/L EDTA and 0.01-0.05% volume fraction Tween-80 or Triton X-100;

the concentration of the proteinase K is 10-30 mg/mL.

2. The kit of claim 1, wherein the lysis solution consists of 0.8mol/L guanidinium isothiocyanate, 4mol/L guanidinium hydrochloride, 1.4mol/L sodium iodide, 25mmol/L Tris-acetic acid, 25mmol/L EDTA, volume fraction 10% Tween-80, 600mmol/L NaCl;

and/or the binding solution consists of 0.6mol/L of guanidinium isothiocyanate, 4mol/L of guanidinium hydrochloride, 1.2mol/L of sodium iodide, 25mmol/L of Tris-acetic acid, 25mmol/L of EDTA, 0.05% of PEG6000 by mass volume fraction, 10% of Tween-80 by volume fraction and 45% of isopropanol by volume fraction;

and/or the cleaning solution consists of 3mol/L guanidine hydrochloride, 0.6mol/L sodium iodide, 25mmol/L Tris-acetic acid, 25mmol/L EDTA, 8% Tween-80 in volume fraction and 40% isopropanol in volume fraction;

and/or the washing solution consists of 5mmol/L Tris-HCl, 0.05 percent of Triton X-100 by volume fraction and 80 percent of absolute ethyl alcohol by volume fraction;

and/or the eluent consists of 5mmol/L Tris-HCl, 0.05mmol/L EDTA and 0.03% Triton X-100 by volume fraction;

and/or the concentration of the proteinase K is 20 mg/mL.

3. The kit of claim 1, wherein the magnetic beads are silica hydroxyl modified magnetic beads with a particle size of 1-2 μm; preferably, the particle size is 2 μm.

4. A lysis solution, binding solution, washing solution and/or elution solution as claimed in claim 1 or 2.

5. Use of a kit according to any one of claims 1 to 3 for the extraction of plasma free DNA.

6. Use of the lysis solution, binding solution, washing solution and/or elution solution of claim 4 for the extraction of free DNA from plasma.

7. A method for extracting plasma free DNA, wherein the method is performed by using the kit of any one of claims 1 to 3, and comprises the following steps:

uniformly mixing protease K, plasma and lysis solution, and incubating to obtain a lysis mixture;

adding the binding solution and the magnetic beads into the cracking mixture after ice bath, uniformly mixing, and standing to obtain a binding mixture;

placing the combined mixture on a magnetic frame for magnetic attraction, removing the supernatant, adding a cleaning solution, uniformly mixing, placing on the magnetic frame for magnetic attraction, and removing the supernatant;

adding a cleaning solution, uniformly mixing, placing on a magnetic frame for magnetic attraction, and then discarding the supernatant;

and (3) drying after centrifugal treatment, adding eluent, centrifuging after shaking, placing on a magnetic frame for magnetic attraction, and absorbing liquid to obtain plasma free DNA.

8. The method of claim 7, wherein the volume ratio of the plasma to the lysis solution, the binding solution, the washing solution, the elution solution, the magnetic beads and the proteinase K is: 1500-2500: 150-250: 2000-2500: 800-120: 800-1000: 30-70: 60-100: 30-50 parts of; preferably, the volume ratio of the blood plasma to the lysis solution, the binding solution, the cleaning solution, the washing solution, the eluent, the magnetic beads and the proteinase K is 2000: 200: 2500: 1000: 1000: 50: 80: 40.

9. plasma free DNA extracted by the method of claim 7 or 8.

10. Use of the plasma-free DNA according to claim 9 for library construction.

Technical Field

The invention relates to the technical field of extraction of free DNA of blood plasma. More particularly, it relates to a kit and a method for extracting plasma free DNA.

Background

Plasma free DNA, Cell-free DNA, refers to the partially degraded collective endogenous DNA in plasma free from cells, mainly derived from apoptosis (Snyder M W, Kircher M, Hill A J, et al, Cell-free DNA complexes in vivo nucleosome gene expression tissues-of-origin [ J ] Cell,2016,164(1-2):57-68.), and the fragment size is concentrated in 150-200 bp. With the intensive research on cfDNA, it was found that cfDNA contains very important genetic information. Because cfDNA and important genetic information (Volik S, Alcalide M, Morin R D, et al. cell-free DNA) contained in cfDNA can be conveniently and non-invasively obtained, and the liquid biopsy technology represented by cfDNA becomes a very important technology for Molecular diagnosis or auxiliary diagnosis, the method is widely applied to the fields of prenatal screening, early Cancer screening, gene mutation detection, Cancer medication guidance, real-time Cancer monitoring and the like.

Methods for extracting cfDNA mainly include a silica gel column method, a magnetic bead method and a phenol chloroform extraction method. Wherein, the phenol chloroform extraction method is basically eliminated because the used reagent is toxic and inconvenient to operate. The silica gel column method and the magnetic bead method are the main methods for extracting the plasma cfDNA on the market at present. The silica gel column extraction has the disadvantages that the column needs to be centrifuged for many times when large-volume plasma is extracted, is very inconvenient, and has the advantage of high extraction quality (no large fragment residue). The magnetic bead method has an advantage in that it can be applied to extraction of large-volume plasma cfDNA, but has a disadvantage in that there is often a problem of low quality (large fragment remains).

Currently, library construction and next generation sequencing of cfDNA are one of the major downstream applications. However, the quality of cfDNA extracted by the magnetic bead method is low (large fragment of DNA remains), so that the problem of low library construction efficiency is generally existed in library construction, and the quality of sequencing data is low, which also limits the accuracy and application range in second-generation sequencing.

Therefore, there is a need to provide a new reagent and method for extracting cfDNA to solve the above problems.

Disclosure of Invention

The first purpose of the invention is to provide a kit for extracting plasma free DNA (cfDNA), which has high quality of extracted cfDNA and can improve the efficiency, the sequencing data quality and the accuracy when being used for constructing a second-generation sequencing library.

The second purpose of the invention is to provide the application of the kit in extracting plasma free DNA (cfDNA).

The third purpose of the invention is to provide a method for extracting plasma free DNA (cfDNA) by using the kit.

The fourth purpose of the invention is to provide the plasma free DNA extracted by the method and the application thereof.

In order to achieve the above object, the present invention firstly provides a kit for extracting plasma free DNA, comprising a Lysis Buffer, a Binding Buffer, a cleaning solution, a Wash Buffer, an eluent, a magnetic bead and a protease K;

the lysis solution contains at least two of 2-6mol/L guanidine hydrochloride, 0.5-1.2mol/L guanidine isothiocyanate and 0.5-2.4mol/L sodium iodide, 10-100mmol/L Tris-acetic acid, 10-100mmol/L EDTA, at least one of Tween-80 and NP-40 with volume fraction of 5-15% (V/V), and 400-800mmol/L NaCl;

the binding solution contains at least two of 2-6mol/L guanidine hydrochloride, 0.5-1.6mol/L guanidine isothiocyanate and 0.5-1.5mol/L sodium iodide, 10-100mmol/L Tris-acetic acid, 10-100mmol/L EDTA, 0.05-0.5% of PEG6000 by mass volume fraction (W/V), 10-15% of at least one of Tween-80 and NP-40 by volume fraction (V/V), and 30-70% of isopropanol by volume fraction (V/V);

the cleaning solution contains at least two of 2-6mol/L guanidine hydrochloride, 0.6-0.8mol/L guanidine isothiocyanate and 0.6-0.8mol/L sodium iodide, 10-100mmol/L Tris-acetic acid, 10-50mmol/L EDTA, at least one of 8-10% (V/V) volume fraction Tween-80 and NP-40 volume fraction, and 30-50% (V/V) volume fraction isopropanol;

the washing solution contains 5-20mmol/L Tris-acetic acid, 0.01-0.05% (V/V) volume fraction of Triton X-100 or Tween-80, and 80% (V/V) volume fraction of absolute ethyl alcohol;

the eluent contains 5-10mmol/L Tris-acetic acid, 0.05-0.3mmol/L EDTA and 0.01-0.05% (V/V) volume fraction Tween-80 or Triton X-100;

the concentration of the proteinase K is 10-30 mg/mL.

Further, the magnetic beads are silicon hydroxyl modified magnetic beads, and the particle size is 1-2 microns; preferably, the particle size is 2 μm.

Preferably, the lysis solution consists of 0.8mol/L guanidine isothiocyanate, 4mol/L guanidine hydrochloride, 1.4mol/L sodium iodide, 25mmol/L Tris-acetic acid, 25mmol/L EDTA, 10% Tween-80 in volume fraction, and 600mmol/L NaCl.

Preferably, the binding solution consists of 0.6mol/L of guanidinium isothiocyanate, 4mol/L of guanidinium hydrochloride, 1.2mol/L of sodium iodide, 25mmol/L of Tris-acetic acid, 25mmol/L of EDTA, 0.05% by volume of PEG6000, 10% by volume of Tween-80 and 45% by volume of isopropanol.

Preferably, the cleaning solution consists of 3mol/L guanidine hydrochloride, 0.6mol/L sodium iodide, 25mmol/L Tris-acetic acid, 25mmol/L EDTA, 8% Tween-80 in volume fraction and 40% isopropanol in volume fraction.

Preferably, the washing solution consists of 5mmol/L Tris-HCl, 0.05 percent by volume of Triton X-100 and 80 percent by volume of absolute ethyl alcohol.

Preferably, the eluent is composed of 5mmol/L Tris-HCl, 0.05mmol/L EDTA, volume fraction 0.03% Triton X-100.

Preferably, the concentration of proteinase K is 20 mg/mL.

The lysis solution, the binding solution, the cleaning solution, the washing solution and/or the eluent in the kit are also within the protection scope of the invention. The kit actually optimizes the lysis solution, the binding solution, the cleaning solution, the washing solution and the eluent respectively, so that the lysis solution, the binding solution, the cleaning solution, the washing solution and the eluent can be independently combined with the existing reagent for extracting the free DNA of the blood plasma by the paramagnetic particle method respectively to extract the free DNA of the blood plasma, thereby improving the extraction quality.

The invention further provides the application of the kit or the lysis solution, the binding solution, the cleaning solution, the washing solution and/or the eluent in the kit in the extraction of the free DNA of the blood plasma.

The kit is developed aiming at extracting cfDNA in blood plasma, so that lysis solution, binding solution, cleaning solution, washing solution and eluent in the kit can independently extract free DNA in blood plasma.

The invention also provides a method for extracting plasma free DNA, which is to extract by using the kit and specifically comprises the following steps:

uniformly mixing protease K, plasma and lysis solution, and incubating to obtain a lysis mixture;

adding the binding solution and the magnetic beads into the cracking mixture after ice bath, uniformly mixing, and standing to obtain a binding mixture;

placing the combined mixture on a magnetic frame for magnetic attraction, removing the supernatant, adding a cleaning solution, uniformly mixing, placing on the magnetic frame for magnetic attraction, and removing the supernatant;

adding a cleaning solution, uniformly mixing, placing on a magnetic frame for magnetic attraction, and then discarding the supernatant;

and (3) drying after centrifugal treatment, adding eluent, centrifuging after shaking, placing on a magnetic frame for magnetic attraction, and absorbing liquid to obtain plasma free DNA.

Further, the volume ratio of the blood plasma to the lysis solution, the binding solution, the cleaning solution, the washing solution, the eluent, the magnetic beads and the proteinase K is as follows: 1500-2500: 150-250: 2000-2500: 800-120: 800-1000: 30-70: 60-100: 30-50 parts of; preferably, the volume ratio of the blood plasma to the lysis solution, the binding solution, the cleaning solution, the washing solution, the eluent, the magnetic beads and the proteinase K is 2000: 200: 2500: 1000: 1000: 50: 80: 40.

further, after the cleaning solution is added and mixed uniformly, the mixture is placed on a magnetic frame for magnetic attraction, the supernatant is discarded, the step is continuously repeated, and then the next step is carried out.

Further, after the washing solution is added and mixed uniformly, the mixture is placed on a magnetic frame for magnetic attraction, the supernatant is discarded, the step is continuously repeated, and then the next step is carried out.

In a specific embodiment of the present invention, the method for extracting plasma free DNA comprises the following steps:

uniformly mixing protease K, plasma and lysis solution by vortex, incubating in a water bath at 60 deg.C for 20min, and shaking for 2-3 times to obtain lysis mixture;

and (3) carrying out ice bath on the cleavage mixture for 5min, adding the binding solution and the magnetic beads, shaking, uniformly mixing, standing at room temperature for 10min, shaking, uniformly mixing for 2-3 times during the period, and obtaining a binding mixture:

placing the combined mixture on a magnetic frame for magnetic attraction for 3min, discarding the supernatant, adding cleaning solution, vortex mixing for 15s, placing on the magnetic frame for magnetic attraction for 1min after moving out, and discarding the supernatant;

adding the cleaning solution again, mixing uniformly for 15s in a vortex manner, magnetically sucking on a magnetic rack for 1min, and discarding the supernatant;

adding washing solution, vortex mixing for 15s, magnetically attracting for 1min on a magnetic frame, and discarding the supernatant;

adding the cleaning solution again, mixing uniformly for 15s by vortex, magnetically sucking on a magnetic rack for 1min, and discarding the supernatant;

centrifuging, discarding the residual liquid, standing for 10min at room temperature, adding the eluent, performing vortex oscillation at room temperature for 5min, centrifuging, magnetically attracting on a magnetic rack for 1min, and absorbing the liquid to obtain cfDNA.

In a more specific embodiment of the present invention, the method for extracting plasma-free DNA comprises the steps of:

uniformly mixing 80 mu L of proteinase K, 2mL of blood plasma and 200 mu L of lysate by vortex, placing the mixture in a water bath kettle at 60 ℃ for incubation for 20min, and uniformly mixing for 2-3 times by oscillation during incubation to obtain a lysis mixture;

and (3) carrying out ice bath on the cleavage mixture for 5min, adding 2.5mL of binding solution and 40 mu L of magnetic beads, shaking and uniformly mixing, standing at room temperature for 10min, shaking and uniformly mixing for 2-3 times during the period to obtain a binding mixture:

placing the combined mixture on a magnetic frame for magnetic attraction for 3min, discarding the supernatant, then adding 1mL of cleaning solution, performing vortex mixing for 15s, removing the mixture, placing the mixture on the magnetic frame for magnetic attraction for 1min, and discarding the supernatant;

adding 1mL of cleaning solution again, mixing uniformly for 15s in a vortex mode, magnetically sucking on a magnetic rack for 1min, and discarding the supernatant;

adding 1mL of washing solution, vortex and uniformly mixing for 15s, magnetically attracting for 1min by using a magnetic frame, and discarding the supernatant;

adding 1mL of washing solution again, mixing uniformly for 15s in a vortex mode, magnetically sucking on a magnetic rack for 1min, and discarding the supernatant;

centrifuging, discarding the residual liquid, standing for 10min at room temperature, air drying, adding 50 μ L of eluent, vortex shaking at room temperature for 5min, centrifuging, magnetically attracting on a magnetic frame for 1min, and absorbing the liquid to obtain cfDNA.

The cfDNA extracted by the method and the application of the cfDNA in library construction are also within the protection scope of the invention.

The invention has the following beneficial effects:

the invention provides a kit and a method for extracting cfDNA from blood plasma by an optimized paramagnetic particle method, which are characterized in that the kit is combined with the cfDNA to the maximum extent and impurities are removed by optimizing a cracking solution, a combined solution, a cleaning solution, a washing solution and an eluent in the kit, particularly optimizing the combination of various guanidine salts in the cracking solution, the combined solution and the cleaning solution and the concentration ratio of a detergent to isopropanol, so that the extracted cfDNA has no large-fragment-degree residue and higher purity, and the kit has higher library construction efficiency, higher sequencing data quality and higher accuracy in a downstream library construction experiment.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

Fig. 1 shows a pattern of peaks of cfDNA obtained in example 6 detected using Agilent 2100; wherein, a, b, c, d, e represent the peak patterns of cfDNA numbered 1, 2, 3, 4, 5, respectively.

Fig. 2 shows a peak pattern diagram of cfDNA obtained by detecting a comparative example using Agilent 2100; wherein, a, B, C, D, e represent peak patterns of cfDNA numbered CQ, A, B, C, D, respectively.

Fig. 3 shows a library peak pattern diagram for detecting cfDNA obtained in example 6 using Agilent 2100; wherein, a, b, c, d, e represent library peak patterns of cfDNA numbered 1, 2, 3, 4, 5, respectively.

FIG. 4 shows library peak patterns of cfDNA obtained from comparative examples detected using Agilent 2100; wherein, a, B, C, D, e represent library peak patterns of cfDNA numbered CQ, A, B, C, D, respectively.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Example 1A kit for extracting plasma free DNA (cfDNA)

A kit for extracting cfDNA from blood plasma by optimizing a magnetic bead method comprises a Lysis solution (Lysis Buffer), a Binding solution (Binding Buffer), a cleaning solution (Clean Buffer), a washing solution (Wash Buffer), an eluent (Elution Buffer), magnetic beads and proteinase K;

wherein the lysine Buffer consists of 0.8mol/L guanidinium isothiocyanate, 4mol/L guanidinium hydrochloride, 1.4mol/L NaI, 25mmol/L Tris-acetic acid, 25mmol/L EDTA, 10 percent (V/V) volume fraction Tween-80 and 600mmol/L NaCl;

the Binding Buffer consists of 0.6mol/L of guanidinium isothiocyanate, 4mol/L of guanidinium hydrochloride, 1.2mol/L of NaI, 25mmol/L of Tris-acetic acid, 25mmol/L of EDTA, 0.05 percent of PEG6000 by mass volume fraction (W/V), 10 percent of Tween-80 by volume fraction (V/V) and 45 percent of isopropanol by volume fraction (V/V);

the Clean Buffer consists of 3mol/L guanidine hydrochloride, 0.6mol/L NaI, 25mmol/L Tris-acetic acid, 25mmol/L EDTA, 8% (V/V) volume fraction Tween-80 and 40% (V/V) volume fraction isopropanol;

the Wash Buffer consists of 5mmol/L Tris-HCl, 0.05 percent (V/V) Triton X-100 of volume fraction and 80 percent (V/V) absolute ethyl alcohol of volume fraction;

the Elution Buffer consists of 5mmol/L Tris-HCl, 0.05mmol/L EDTA and 0.03% (V/V) volume fraction Triton X-100;

the magnetic beads are silicon hydroxyl modified magnetic beads, and the particle size is 2 mu m;

the concentration of proteinase K was 20 mg/mL.

Example 2 kit for extracting cfDNA

A kit for extracting cfDNA from blood plasma by optimizing a magnetic bead method comprises a Lysis solution (Lysis Buffer), a Binding solution (Binding Buffer), a cleaning solution (Clean Buffer), a washing solution (Wash Buffer), an eluent (Elution Buffer), magnetic beads and proteinase K;

wherein the lysine Buffer consists of 2mol/L guanidine hydrochloride, 1.2mol/L guanidine isothiocyanate and 2.4mol/L NaI, 100mmol/L Tris-acetic acid, 100mmol/L EDTA, 15% (V/V) Tween-80 and 800mmol/L NaCl;

the Binding Buffer consists of 2mol/L guanidine hydrochloride, 1.6mol/L guanidine isothiocyanate, 1.5mol/L NaI, 100mmol/L Tris-acetic acid, 100mmol/L EDTA, 0.5% (W/V) PEG6000, 15% (V/V) Tween-80 and 70% (V/V) isopropanol;

the Clean Buffer consists of 2mol/L guanidine hydrochloride, 0.8mol/L guanidine isothiocyanate, 100mmol/L Tris-acetic acid, 50mmol/L EDTA, 10% (V/V) Tween-80 and 50% (V/V) isopropanol;

the Wash Buffer consists of 20mmol/L Tris-acetic acid, 0.05% (V/V) Tween-80 and 80% (V/V) absolute ethanol;

elution Buffer consists of 10mmol/L Tris-acetic acid, 0.1mmol/L EDTA, and 0.01% (V/V) Tween-80;

the magnetic beads are silicon hydroxyl modified magnetic beads, and the particle size is 2 mu m;

the concentration of proteinase K was 10 mg/mL.

Example 3 a kit for extracting cfDNA

A kit for extracting cfDNA from blood plasma by optimizing a magnetic bead method comprises a Lysis solution (Lysis Buffer), a Binding solution (Binding Buffer), a cleaning solution (Clean Buffer), a washing solution (Wash Buffer), an eluent (Elution Buffer), magnetic beads and proteinase K;

wherein the lysine Buffer consists of 6mol/L guanidine hydrochloride, 0.5mol/L guanidine isothiocyanate, 0.5mol/L NaI, 10mmol/L Tris-acetic acid, 10mmol/L EDTA, 5% (V/V) NP-40, and 400mmol/L NaCl;

the Binding Buffer consists of 6mol/L guanidine hydrochloride, 0.5mol/L guanidine isothiocyanate, 0.5mol/L NaI, 10mmol/L Tris-acetic acid, 10mmol/L EDTA, 0.05% (W/V) PEG6000, 10% (V/V) NP-40, and 30% (V/V) isopropanol;

the Clean Buffer consists of 6mol/L guanidine hydrochloride, 0.6mol/L NaI, 10mmol/L Tris-acetic acid, 10mmol/L EDTA, 10% (V/V) NP-40, and 30% (V/V) isopropanol;

wash Buffer consisted of 5mmol/L Tris-acetate, 0.05% (V/V) Triton X-100, and 80% (V/V) absolute ethanol;

elution Buffer consists of 5mmol/L Tris-acetic acid, 0.3mmol/L EDTA, and 0.05% (V/V) Triton X-100;

the magnetic beads are silicon hydroxyl modified magnetic beads, and the particle size is 1 mu m;

the concentration of proteinase K was 20 mg/mL.

Example 4 a kit for extracting cfDNA

A kit for extracting cfDNA from blood plasma by optimizing a magnetic bead method comprises a Lysis solution (Lysis Buffer), a Binding solution (Binding Buffer), a cleaning solution (Clean Buffer), a washing solution (Wash Buffer), an eluent (Elution Buffer), magnetic beads and proteinase K;

wherein the lysine Buffer consists of 4mol/L guanidine hydrochloride, 1.4mol/L NaI, 50mmol/L Tris-acetic acid, 50mmol/L EDTA, 10% (V/V) Tween-80 and 600mmol/L NaCl;

the Binding Buffer consists of 4mol/L guanidine hydrochloride, 1.6mol/L guanidine isothiocyanate, 50mmol/L Tris-acetic acid, 50mmol/L EDTA, 0.5% (W/V) PEG6000, 15% (V/V) Tween-80 and 70% (V/V) isopropanol;

the Clean Buffer consists of 3mol/L guanidine hydrochloride, 0.6mol/L guanidine isothiocyanate, 25mmol/L Tris-acetic acid, 25mmol/L EDTA, 8% (V/V) Tween-80 and 40% (V/V) isopropanol;

the Wash Buffer consists of 10mmol/L Tris-acetic acid, 0.01% (V/V) Tween-80 and 80% (V/V) absolute ethanol;

the Elution Buffer consists of 10mmol/L Tris-acetic acid, 0.1mmol/L EDTA and 0.01% (V/V) Tween-80;

the magnetic beads are silicon hydroxyl modified magnetic beads, and the particle size is 1 mu m;

the concentration of proteinase K was 30 mg/mL.

Example 5A kit for extracting cfDNA

A kit for extracting cfDNA from blood plasma by optimizing a magnetic bead method comprises a Lysis solution (Lysis Buffer), a Binding solution (Binding Buffer), a cleaning solution (Clean Buffer), a washing solution (Wash Buffer), an eluent (Elution Buffer), magnetic beads and proteinase K;

wherein the lysine Buffer consists of 0.8mol/L guanidinium isothiocyanate, 2.4mol/L NaI, 50mmol/L Tris-acetic acid, 50mmol/L EDTA, 10% (V/V) Tween-80 and 600mmol/L NaCl;

the Binding Buffer consists of 4mol/L guanidine hydrochloride, 1.5mol/L NaI, 50mmol/L Tris-acetic acid, 50mmol/L EDTA, 0.5% (W/V) PEG6000, 10% (V/V) NP-40 and 70% (V/V) isopropanol;

the Clean Buffer consists of 3mol/L guanidine hydrochloride, 0.8mol/L NaI, 25mmol/L Tris-acetic acid, 25mmol/L EDTA, 8% (V/V) NP-40, and 40% (V/V) isopropanol;

the Wash Buffer consists of 5mmol/L Tris-acetic acid, 0.01% (V/V) Tween-80 and 80% (V/V) absolute ethanol;

the Elution Buffer consists of 5mmol/L Tris-acetic acid, 0.1mmol/L EDTA, and 0.01% (V/V) Triton X-100;

the magnetic beads are silicon hydroxyl modified magnetic beads, and the particle size is 2 mu m;

the concentration of proteinase K was 20 mg/mL.

Example 6 extraction of cfDNA Using the kits of examples 1-5

cfDNA extraction was performed on 2ml of plasma (consistent amount of each Buffer) using the kits of examples 1-5, respectively, comprising the following steps:

adding 80 mu L of proteinase K, 2mL of blood plasma and 200 mu L of lysine Buffer into a 15mL centrifuge tube, uniformly mixing by vortex, placing the mixture in a water bath kettle at 60 ℃ for incubation for 20min, and uniformly mixing by shaking for 3 times during the incubation to obtain a Lysis mixture;

the mixture is subjected to ice bath for 5min, then 2.5mL Binding Buffer and 40 mu L magnetic beads are added into a centrifuge tube, the mixture is stirred and mixed evenly and then stands for 10min at room temperature, and the mixture is stirred and mixed evenly for 3 times during the period to obtain a Binding mixture;

placing the centrifuge tube on a 15mL magnetic frame for magnetic absorption for 3min, discarding the supernatant, then adding 1mL Clean Buffer, uniformly mixing by vortex for 15s, moving the mixture into a new 1.5mL centrifuge tube, placing the centrifuge tube on the magnetic frame for magnetic absorption for 1min, and discarding the supernatant;

adding 1mL of Clean Buffer again, mixing uniformly for 15s in a vortex mode, magnetically attracting for 1min by using a magnetic rack, and discarding the supernatant;

adding 1mL of Wash Buffer, uniformly mixing for 15s in a vortex mode, magnetically attracting for 1min by using a magnetic rack, and discarding the supernatant;

adding 1mL of Wash Buffer again, mixing uniformly for 15s in a vortex mode, magnetically attracting for 1min by using a magnetic rack, and discarding the supernatant;

and (3) after short-time centrifugation (less than 3s), discarding the residual liquid, uncapping at room temperature, standing for 10min for air drying, adding 40 mu L of precipitation Buffer, carrying out vortex shaking at room temperature for 5min, after short-time centrifugation (less than 3s), magnetically attracting for 1min by using a magnetic rack, and sucking the liquid into a new 1.5mL centrifuge tube to obtain cfDNA. cfDNA extracted from the kit of examples 1-5 was numbered 1, 2, 3, 4 and 5, respectively, and stored at-20 ℃ or subjected to downstream experiments (concentration determination and library construction).

Comparative example 1 Company Q cfDNA extraction kit extraction cfDNA

cfDNA was extracted from 2mL of plasma using the cfDNA extraction kit from Company Q (Cat. No.: 55114) according to the protocol to give cfDNA, numbered CQ.

Comparative example 2 extraction of cfDNA with silica gel column of Company Q in combination with the kit of example 1

The cfDNA was extracted by replacing the magnetic beads in the kit of example 1 with silica gel column (catalog No. 55114) of Company Q, comprising the following steps:

adding 80 mu L of proteinase K, 2mL of blood plasma and 200 mu L of lysine Buffer into a 15mL centrifuge tube, uniformly mixing by vortex, placing in a water bath kettle at 60 ℃ for incubation for 20min, and uniformly mixing by shaking for 3 times in the period;

after cracking, carrying out ice bath for 3-5min, then adding 2.5mL Binding Buffer into the tube, shaking and uniformly mixing, centrifuging for 10min at 12,000g/min, adding the supernatant into a Q company silica gel column for multiple times, centrifuging for 5min at 5,000g/min, and discarding flow through:

adding 1mL of Clean Buffer into the silica gel column, standing for 15s, centrifuging for 1min at 5,000g/min, and discarding flow-through;

adding 1mL of Clean Buffer into the silica gel column again, standing for 15s, centrifuging for 1min at 5,000g/min, and discarding flow-through;

adding 1mL of Wash Buffer into the silica gel column, standing for 15s, centrifuging for 1min at 5,000g/min, and discarding the flow-through;

adding 1mL of Wash Buffer into the silica gel column again, standing for 15s, centrifuging for 1min at 5,000g/min, and discarding the breakthrough;

centrifuging silica gel column for 2min at 5000g/min, discarding the residual liquid and collecting tube, placing in a new centrifuge tube, uncapping the silica gel column at room temperature, standing for 5-10min to volatilize ethanol, adding 30-50 μ L of precipitation Buffer onto the silica gel column inner membrane, standing at room temperature for 5min, centrifuging for 1min at >12,000g/min, sucking liquid into a new centrifuge tube of 1.5mL to obtain cfDNA (number Q), and placing at-20 ℃ for preservation or performing downstream experiments (concentration determination and library construction).

Comparative example 3 kit and method for extracting cfDNA

A kit for extracting cfDNA comprises lysis solution 1, binding solution 1, cleaning solution 1, eluent 1, magnetic beads and proteinase K;

wherein, the lysate 1 consists of 4mol/L guanidinium isothiocyanate, 50mmol/L Tris-HCl, 25mmol/L LEDTA, 12% (V/V) Triton X-100 and 300mmol/L NaCl;

binding solution 1 consists of 3.5mol/L guanidinium isothiocyanate, 50mmol/L Tris-HCl, 25mmol/L EDTA, 12% (V/V) Triton X-100 and 30% (V/V) isopropanol;

cleaning solution 1 consists of 2mol/L guanidinium isothiocyanate, 50mmol/L Tris-HCl, 25mmol/L EDTA, 5% (V/V) Triton X-100 and 30% (V/V) isopropanol;

washing solution 1 was prepared from: 10mmol/L Tris-HCl and 80% (V/V) absolute ethyl alcohol;

eluent 1 is composed of nucleic-free water;

the magnetic beads are silicon hydroxyl modified magnetic beads, and the particle size is 2 mu m;

the concentration of proteinase K was 20 mg/mL.

A method for extracting cfDNA by using the kit comprises the following steps:

1.1, sequentially adding 80 mu L of proteinase K, 2mL of plasma and 200 mu L of lysate 1 into a 15mL BD tube, oscillating and uniformly mixing, and then incubating for 20min at 60 ℃;

1.2, carrying out ice bath for 5min, adding 2.5mL of binding solution 1 and 40 mu L of magnetic beads, and oscillating for 10min for 3 times;

1.3 placing on a magnetic frame for 3min, discarding the supernatant, adding 1mL of cleaning solution 1, and shaking for 15 s;

1.4 completely transferring the magnetic bead solution into a 1.5ml centrifuge tube, and placing the centrifuge tube in a magnetic frame for 1 min;

1.5, abandoning the supernatant, adding 1mL of cleaning solution 1, shaking and uniformly mixing for 15s, and placing on a magnetic frame for 1 min;

1.6, discarding the supernatant, adding 1ml of washing solution 1, shaking and uniformly mixing for 15s, and placing on a magnetic frame for 1 min;

1.7, discarding the supernatant, adding 1ml of washing solution 1, shaking and uniformly mixing for 15s, and placing on a magnetic frame for 1 min;

1.8 discarding the supernatant, air-drying at room temperature for 10min, adding 40 μ L of eluent 1, and oscillating at room temperature for 5 min;

1.9 placing on a magnetic frame for 1min, sucking liquid into a new centrifuge tube to obtain cfDNA, numbering A, and storing at-20 ℃ for detection (concentration determination and library construction).

Comparative example 4 kit and method for extracting cfDNA

A kit for extracting cfDNA comprises lysis solution 2, binding solution 2, cleaning solution 2, eluent 2, a silica gel adsorption column and proteinase K;

wherein, the lysate 2 consists of 6mol/L guanidinium isothiocyanate, 100mmol/L Tris-HCl, 20mmol/L LEDTA, 10% (V/V) SDS buffer solution, 300mmol/L NaCl and 1% (V/V) DMSO;

binding solution 2 consisted of 5mol/L guanidinium isothiocyanate, 3mol/L thiourea, 1mol/L NaCl, 50mmol/L Tris-HCl, 20% (W/V) PEG6000, 3.5% (W/V) sodium citrate, 20mmol/L HEPS-KOH, 1% (V/V) Triton X-100 and 20% (V/V) isopropanol (added before use);

the cleaning solution 2 consists of 100mmol/L Tris-HCl, 6mol/L guanidine hydrochloride, 1mol/L NaCl, 10mmol/LEDTA and 60% (V/V) absolute ethyl alcohol;

washing solution 2 was prepared from: 100mmol/L Tris-HCl and 80% (V/V) absolute ethyl alcohol;

eluent 2 is composed of 5mmol/L Tris-HCl;

the silica gel adsorption column was obtained from 55114 from Company Q.

The concentration of proteinase K was 20 mg/mL.

A method for extracting cfDNA by using the kit comprises the following steps:

2.1 centrifuging 2.4ml of plasma and 4500g for 10min, taking supernatant, centrifuging at 15000g for 10min, and removing 2ml of supernatant;

2.2 adding 80 mul protease K and 200 mul lysate 2 into the supernatant, shaking and mixing evenly for 30s, and incubating for 30min at 60 ℃;

2.3 add 2.5mL of binding solution 2 to the lysate; shaking and mixing uniformly for 30 s; performing ice bath for 5 min; adding the product into silica gel adsorption column, centrifuging at 10000g for 30s, and removing the flow-through until all the product flows through the silica gel column;

2.4 adding 1mL of cleaning solution 2 into the adsorption column; 10000g of centrifugation for 30s and discarding the flow-through.

2.5 adding 1mL of washing solution 2 into the adsorption column; 10000g of centrifugation for 30s and discarding the flow-through.

2.6 adding 1mL of washing solution 2 into the adsorption column; 10000g of centrifugation for 30s and discarding the flow-through.

2.7 centrifuging the adsorption column at 10000g for 2 min; the adsorption column was transferred to a new collection tube.

2.8 adding 40 μ L of eluent 2 into the adsorption column; standing at room temperature for 3 min.

2.9 centrifuging the column at 10000g for 2min, collecting the liquid to obtain cfDNA, numbering B, and storing at-20 deg.C for detection (concentration determination and library construction).

Comparative example 5 kit and method for extracting cfDNA

A kit for extracting cfDNA, comprising a lysis solution and a binding solution in example 1, and a cleaning solution 1, a washing solution 1, an eluent 1, magnetic beads and proteinase K in comparative example 3;

wherein the lysis solution consists of 0.8mol/L guanidinium isothiocyanate, 4mol/L guanidinium hydrochloride, 1.4mol/L NaI, 25mmol/L Tris-acetic acid, 25mmol/L EDTA, 10% (V/V) Tween-80 and 600mmol/L NaCl;

the binding solution consists of 0.6mol/L of guanidinium isothiocyanate, 4mol/L of guanidinium hydrochloride, 1.2mol/L of NaI, 25mmol/L of Tris-acetic acid, 25mmol/L of EDTA, 0.05% (W/V) of PEG6000, 10% (V/V) of Tween-80 and 45% (V/V) of isopropanol;

cleaning solution 1 consists of 2mol/L guanidinium isothiocyanate, 50mmol/L Tris-HCl, 25mmol/L EDTA, 5% (V/V) Triton X-100 and 30% (V/V) isopropanol;

washing solution 1 consists of 10mmol/L Tris-HCl and 80% (V/V) absolute ethyl alcohol;

eluent 1 is composed of nucleic-free water;

the magnetic beads are silicon hydroxyl modified magnetic beads, and the particle size is 2 mu m;

the concentration of proteinase K was 20 mg/mL.

A method for extracting cfDNA by using the kit comprises the following steps:

3.1 sequentially adding 80 mu L of proteinase K, 2mL of plasma and 200 mu L of lysate into a 15mL BD tube, oscillating and uniformly mixing, and then incubating for 20min at 60 ℃;

3.2, carrying out ice bath for 5min, adding 2.5mL of binding solution and 40 mu L of magnetic beads, and oscillating for 10min for 3 times;

3.3 placing on a magnetic frame for 3min, discarding the supernatant, adding 1mL of cleaning solution 1, and shaking for 15 s;

3.4 completely transferring the magnetic bead solution into a 1.5ml centrifuge tube, and placing the centrifuge tube in a magnetic frame for 1 min;

3.5, abandoning the supernatant, adding 1mL of cleaning solution 1, shaking and uniformly mixing for 15s, and placing on a magnetic frame for 1 min;

3.6 abandoning the supernatant, adding 1ml of washing solution 1, shaking and uniformly mixing for 15s, and placing on a magnetic frame for 1 min;

3.7, discarding the supernatant, adding 1ml of washing solution 1, shaking and uniformly mixing for 15s, and placing on a magnetic frame for 1 min;

3.8 abandoning the supernatant, drying in the air for 10min at room temperature, adding 40 mu L of eluent 1, and oscillating for 5min at room temperature;

3.9 placing on a magnetic frame for 1min, sucking liquid into a new centrifuge tube to obtain cfDNA, numbering C, and storing at-20 ℃ for detection (concentration determination and library construction).

Comparative example 6 kit and method for extracting cfDNA

A kit for extracting cfDNA comprises lysis solution 1 and binding solution 1 in comparative example 3, and a cleaning solution, a washing solution, an eluent, magnetic beads and proteinase K in example 1;

wherein, the lysate 1 consists of 4mol/L guanidinium isothiocyanate, 50mmol/L Tris-HCl, 25mmol/L LEDTA, 12% (V/V) Triton X-100 and 300mmol/L NaCl;

binding solution 1 consists of 3.5mol/L guanidinium isothiocyanate, 50mmol/L Tris-HCl, 25mmol/L EDTA, 12% (V/V) Triton X-100 and 30% (V/V) isopropanol;

the cleaning solution consists of 3mol/L guanidine hydrochloride, 0.6mol/L NaI, 25mmol/L Tris-acetic acid, 25mmol/LEDTA, 8% T (V/V) ween-80 and 40% (V/V) isopropanol;

the washing solution consists of 5mmol/L Tris-HCl, 0.05% (V/V) Triton X-100 and 80% (V/V) absolute ethyl alcohol;

the eluent consists of 5mmol/L Tris-HCl, 0.5mmol/L EDTA and 0.03% (V/V) Triton X-100;

the magnetic beads are silicon hydroxyl modified magnetic beads, and the particle size is 2 mu m;

the concentration of proteinase K was 20 mg/mL.

A method for extracting cfDNA by using the kit comprises the following steps:

4.1 adding 80 μ L proteinase K, 2mL blood plasma and 200 μ L lysate into 15mL BD tube in turn, shaking and mixing uniformly, and incubating at 60 deg.C for 20 min;

4.2, carrying out ice bath for 5min, adding 2.5mL of binding solution and 40 mu L of magnetic beads, and oscillating for 10min for 3 times;

4.3 placing on a magnetic frame for 3min, discarding the supernatant, adding 1mL of cleaning solution 1, and shaking for 15 s;

4.4 completely transferring the magnetic bead solution into a 1.5ml centrifuge tube, and placing the centrifuge tube in a magnetic frame for 1 min;

4.5, abandoning the supernatant, adding 1mL of cleaning solution 1, shaking and uniformly mixing for 15s, and placing on a magnetic frame for 1 min;

4.6 abandoning the supernatant, adding 1ml of washing solution 1, shaking and uniformly mixing for 15s, and placing on a magnetic frame for 1 min;

4.7 abandoning the supernatant, adding 1ml of washing solution 1, shaking and mixing uniformly for 15s, and placing on a magnetic frame for 1 min;

4.8 abandoning the supernatant, drying in the air for 10min at room temperature, adding 40 mu L of eluent 1, and oscillating for 5min at room temperature;

4.9 placing on a magnetic frame for 1min, sucking liquid into a new centrifuge tube to obtain cfDNA with the number D, and storing at-20 ℃ for detection (concentration determination and library construction).

Experimental example 1 quality test of cfDNA obtained in example 6 and comparative examples 1 to 6

The quality, i.e., concentration, total amount and fragment size, of the cfdnas numbered 1, 2, 3, 4 and 5 extracted in example 6 (i.e., the cfdnas extracted using the kits of examples 1 to 5) and the cfdnas numbered CQ, Q, a, B, C and D obtained in comparative examples 1 to 6 were measured by Qubit 3.0, and the results are shown in table 1, which shows that the extraction of the cfdnas numbered 1, 2, 3, 4 and 5 was normal, while the yield of comparative example 1 was slightly low, and when the cfdnas were extracted by silica gel column with Company Q (comparative example 2), the extraction failed, indicating that the kit of the present invention is not suitable for extraction by silica gel column (not shown in Agilent detection diagram), and all of comparative examples 3 to 6 could extract the cfdnas, but the effect was not good. The results of detecting the peak patterns of cfDNA by using Agilent 2100 are shown in fig. 1 and 2, and the specific results are as follows:

the peak pattern of cfDNA extracted using the kit of example 1 was detected using Agilent 2100, and the result is shown in a in fig. 1, which shows that the peak pattern was most smooth, no large fragments remained, and the extraction was normal.

When the peak pattern of cfDNA extracted by the kit of example 2 was detected by Agilent 2100, the result is shown as b in fig. 1, which shows that no significant large fragment remained and the extraction was normal.

When the peak pattern of cfDNA extracted using the kit of example 3 was detected using Agilent 2100, the result is shown as c in fig. 1, which shows no significant large fragment remaining and normal extraction.

When the peak pattern of cfDNA extracted using the kit of example 4 was detected using Agilent 2100, the result is shown as d in fig. 1, which shows no significant large fragment remaining, normal extraction, but low concentration.

When the peak pattern of cfDNA extracted using the kit of example 5 was detected using Agilent 2100, the result is shown as e in fig. 1, which shows no significant large fragment remaining, normal extraction, but low concentration.

The peak pattern of cfDNA obtained in comparative example 1 was examined using Agilent 2100, and the results are shown in a in fig. 2, showing that extraction was normal, but with a slightly larger fragment tailing, and yield was slightly lower.

The peak pattern of the cfDNA obtained in comparative example 3 was examined using Agilent 2100, and the result is shown in b in fig. 2, which shows that the extraction was normal, but there was a slight large smear and the effect was not good.

The peak pattern of the cfDNA obtained in comparative example 4 was detected by Agilent 2100, and the result is shown in c in fig. 2, which shows that the cfDNA was extracted normally at the main peak (170bp), but had severe large fragment tailing and poor effect.

The peak pattern of the cfDNA obtained in comparative example 5 was detected by Agilent 2100, and the result is shown in d in fig. 2, which shows that the extraction of cfDNA at the main peak (170bp) was normal, but there was severe large fragment tailing, and the effect was not good.

The peak pattern of cfDNA obtained in comparative example 6 was examined using Agilent 2100, and the result is shown as e in fig. 2, showing that extraction was normal, but the concentration was low.

TABLE 1 results of comparing the quality of cfDNA obtained in example 6 and comparative examples 1 to 6

Numbering	Concentration (ng/. mu.l)	Total amount (ng)	Fragment size (bp)
				1	0.788	31.52	176
2	1.145	45.79	179
				3	1.281	51.23	177
4	0.638	25.52	176
				5	0.651	26.04	179
CQ	0.614	24.56	178
				Q	Is almost zero	-	-
A	0.256	10.24	175
				B	1.512	60.48	176
C	1.872	74.88	179
				D	0.561	22.44	178

Test example 2 DNA library construction Using cfDNA obtained in example 6 and comparative examples 1 to 6

DNA library construction was performed by taking cfDNA numbered 1, 2, 3, 4 and 5 extracted in example 6 (i.e., cfDNA extracted using the kit of examples 1-5) and cfDNA numbered CQ, a, B, C and D10 ng (10 cycles) obtained in comparative examples 1, 3, 4, 5 and 6, respectively; the kit used for library construction was from Beijing Quanji Biotechnology Ltd (catalog number: KP201), and the procedure was performed according to the requirements of the kit instructions.

Yield results of the libraries are shown in table 2, while the peak patterns of the cfDNA library detected by Agilent 2100 are shown in fig. 3 and 4, showing that the library yields of numbers 1, 2 and 3 are dominant in yield, the peak patterns are normal, and the concentrations and yields of numbers 4 and 5, CQ and a and D are inferior; while the numbers B and C are high in yield, the peak patterns of the products are seen as large fragment contamination, and the specific peak pattern results are as follows:

the library peak pattern of cfDNA extracted by the kit of example 1 was detected by Agilent 2100, and the result is shown as a in fig. 3, which shows that the extraction was normal and the ratio of the main peak was the highest.

The library peak pattern of cfDNA extracted by the kit of example 2 was detected by Agilent 2100, and the result is shown in b in fig. 3, which shows that the extraction was normal and the ratio of the main peak was superior, slightly lower than that of example 1.

The library peak pattern of cfDNA extracted using the kit of example 3 was examined using Agilent 2100, and the results are shown in c in fig. 3, which shows that extraction was normal, but the proportion of major peaks was lower than in example 1.

The library peak pattern of cfDNA extracted using the kit of example 4 was examined using Agilent 2100, and the results are shown in d in fig. 3, which shows that extraction was normal, but the proportion of major peaks was lower than in example 1.

The library peak pattern of cfDNA extracted using the kit of example 5 was examined using Agilent 2100, and the results are shown as e in fig. 3, which shows that extraction was normal, but the proportion of major peaks was lower than in example 1.

The library peak pattern of the cfDNA obtained in comparative example 1 was examined using Agilent 2100, and the result is shown in a in fig. 4, which shows that extraction was normal, but the proportion of the major peak was lower than in example 1.

The library peak pattern of the cfDNA obtained in comparative example 3 was examined using Agilent 2100, and the result is shown in b in fig. 4, which shows that extraction was normal, but the proportion of the major peak was lower than in example 1.

The library peak pattern of the cfDNA obtained in comparative example 4 was examined using Agilent 2100, and the result is shown in c in fig. 4, which shows that extraction was normal, but the proportion of the major peak was lower than that of example 1, and a large non-specific library fragment appeared.

The library peak pattern of the cfDNA obtained in comparative example 5 was examined using Agilent 2100, and the result is shown in d in fig. 4, which shows that extraction was normal, but the proportion of the major peak was lower than that of example 1, and a large non-specific library fragment appeared.

The library peak pattern of cfDNA obtained in comparative example 6 was examined using Agilent 2100, and the result is shown as e in fig. 4, which shows that extraction was normal, but the proportion of major peaks was lower than in example 1.

TABLE 2 comparison of DNA library construction results of cfDNA obtained in example 6 and comparative examples 1 to 6

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.