Tumor DNA liquid biopsy detection method

Abstract

The invention relates to the field of tumor DNA liquid biopsy, in particular to a tumor DNA liquid biopsy detection method which comprises the steps of sample collection, DNA evaluation, library construction, PCR amplification, sequencing reaction and data collection and data analysis. According to the present invention, the PARP3 enzyme, the sequencing linker not connected with the DNA and the impurities generated during the PCR amplification can be decomposed by using the purification enzyme composition, such that the magnetic bead purification step can be replaced by using the purification enzyme composition in the library construction and PCR amplification steps, and the purification enzyme composition prepared from the trypsin and the UDG enzyme has characteristics of low price, good purification effect, and good purification effect. Moreover, the problem of residual magnetic bead particles does not exist, and the precision of a measurement result can be further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor DNA liquid biopsy, and in particular to a tumor DNA liquid biopsy detection method. Background Art

[0002] Tumor DNA liquid biopsy is an advanced medical testing technology that provides information about tumors by analyzing tumor DNA fragments in patient body fluids. Tumor DNA liquid biopsy involves obtaining a portion of a patient's body fluids (primarily blood) through non-invasive or minimally invasive means. Using advanced technologies such as high-throughput sequencing and digital PCR, the system analyzes circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomes in the fluids to obtain relevant information about the tumor.

[0003] Next-generation sequencing (NGS) is an important technology for tumor DNA liquid biopsy. It uses a high-throughput sequencing platform to simultaneously detect a large number of DNA sequences in a sample, thereby achieving a comprehensive analysis of tumor DNA.

[0004] During the next-generation sequencing process, magnetic bead purification is required to remove the PARP3 enzyme used in library construction, sequencing adapters that are not connected to DNA, and impurities generated during PCR amplification to improve DNA purity. However, magnetic bead purification still has certain disadvantages in practical applications, such as:

[0005] 1. The cost of magnetic bead purification is usually higher than some traditional purification methods. This is mainly due to the high cost of the preparation of the magnetic beads themselves and the related magnetic separation equipment.

[0006] 2. During the magnetic bead purification process, improper operation or poor quality of magnetic beads will result in residual magnetic beads. These residual magnetic beads may interfere with subsequent experiments and affect the accuracy of the results. Summary of the Invention

[0007] The purpose of the present invention is to address the shortcomings of the existing technology and propose a tumor DNA liquid biopsy detection method.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A tumor DNA liquid biopsy detection method, including a tumor DNA liquid biopsy detection method, comprising the following steps:

[0010] S1: sample collection;

[0011] a: Practical venous blood collection is used to collect blood from patients;

[0012] b: Use EDTA anticoagulant tubes to preserve the collected blood;

[0013] c) Place the EDTA anticoagulant tube into a centrifuge to separate the plasma and cell components;

[0014] d: adding proteinase K to the plasma sample and allowing the plasma sample to stand for a period of time;

[0015] e) extracting DNA from the plasma sample using a DNA extraction kit and placing the extracted DNA in a DNA storage tube;

[0016] S2: DNA assessment;

[0017] a: Use gel electrophoresis to detect DNA fragment size and integrity;

[0018] b: DNA purity was measured using spectrophotometry;

[0019] S3: Library construction;

[0020] c) Pour Tn5 transposase, Tris-HCl buffer and DNA sample into a centrifuge tube and gently mix the reaction system;

[0021] d: Incubate the reaction system at an appropriate temperature to allow the enzyme to fully act;

[0022] e) When the DNA is broken to the desired length, ethylenediaminetetraacetic acid is added to the centrifuge tube to inhibit Tn5 transposase;

[0023] f: Add ethanol precipitation and phenol to the centrifuge tube to purify the DNA;

[0024] g: Use PARP3 enzyme to repair the ends of DNA fragments, making them have blunt and sticky ends;

[0025] h: Add sequencing adapters to both ends of the DNA fragments and ensure that the concentration of the adapters matches the concentration of the DNA fragments;

[0026] i: adding a purification enzyme composition into the centrifuge tube to decompose the PARP3 enzyme and the sequencing adapter that is not connected to the DNA, thereby improving the purity of the DNA;

[0027] S4: PCR amplification;

[0028] a: Design specific primers according to the sequence of the adapter;

[0029] b: PCR amplification of the library using a high-fidelity DNA polymerase;

[0030] c: Pour the purified enzyme composition into a centrifuge tube to remove the impurities generated by PCR amplification, further improving the purity of the DNA;

[0031] S5: Sequencing reaction and data collection;

[0032] a: Pretreatment of the sequencing chip, including cleaning, coating and other steps;

[0033] b: Load the amplified library onto the sequencing chip;

[0034] c: Perform sequencing reaction on the sequencer and capture the fluorescence signal at the end of the newly synthesized DNA strand;

[0035] d: Scan the sequencing chip using a laser to collect the fluorescence signal of each DNA cluster;

[0036] S6: Data analysis;

[0037] a: Quality control of the raw data obtained by sequencing, including removing low-quality sequences and removing adapter sequences;

[0038] b: Align the processed sequences with the reference genome to determine the sequence position on the genome;

[0039] c: Use bioinformatics tools to detect variations in the alignment results;

[0040] d: According to the results of variation detection, combined with the clinical information and tumor characteristics of the patient, perform result interpretation and reporting.

[0041] Preferably, the centrifuge speed in step S1-c is 3000 rpm, and the centrifugation time is 10-15 min.

[0042] Preferably, the standing time in step S1-d is 10-14 h, and the plasma sample is placed in a water bath at 55°C.

[0043] Preferably, the purified enzyme composition comprises a mixture of trypsin and UDG enzyme, and the weight ratio of trypsin to UDG enzyme is 2:1.

[0044] Preferably, the trypsin and UDG enzyme are mixed in a blender at a stirring speed of 200 rpm, a stirring temperature of 37°C, and a stirring solution pH of 7.5.

[0045] Preferably, the number of PCR cycles in step S4-b is selected between 25-40, the denaturation temperature is 94-95°C for 2-10 min, and the annealing temperature is 65°C for 30-60 s.

[0046] Preferably, in step S5-b, it is necessary to ensure that the concentration of the library matches the requirements of the sequencing chip.

[0047] Preferably, low-quality sequences and adapter sequences need to be removed in step S6-a.

[0048] Preferably, the data analyzed in step S6-c includes single nucleotide polymorphisms (SNPs), insertions and deletions, and structural variations.

[0049] The beneficial effects of the present invention are as follows: the PARP3 enzyme, sequencing adapters not connected to DNA, and impurities generated during PCR amplification can be decomposed by the purified enzyme composition. In this way, in the library construction and PCR amplification steps, the purified enzyme composition can be used to replace the magnetic bead purification step. The purified enzyme composition prepared by trypsin and UDG enzyme is not only low in price, but also does not have the problem of residual magnetic bead particles, which can further improve the accuracy of the measurement results. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0051] A tumor DNA liquid biopsy detection method comprises the following steps:

[0052] S1: sample collection;

[0053] a: Practical venous blood collection is used to collect blood from patients;

[0054] b: Use EDTA anticoagulant tubes to preserve the collected blood;

[0055] c) Place the EDTA anticoagulant tube into a centrifuge to separate the plasma and cell components;

[0056] d: adding proteinase K to the plasma sample and allowing the plasma sample to stand for a period of time;

[0057] e) extracting DNA from the plasma sample using a DNA extraction kit and placing the extracted DNA in a DNA storage tube;

[0058] S2: DNA assessment;

[0059] a: Use gel electrophoresis to detect DNA fragment size and integrity;

[0060] b: DNA purity was measured using spectrophotometry;

[0061] S3: Library construction;

[0062] c: Pour Tn5 transposase, Tris-HCl buffer and DNA sample into a centrifuge tube and mix gently;

[0063] d: Incubate the reaction at an appropriate temperature to allow the enzyme to work fully;

[0064] e: When the DNA is broken to the desired length, add ethylenediaminetetraacetic acid to the centrifuge tube to inhibit the Tn5 transposase;

[0065] f: Add ethanol precipitation and phenol to the centrifuge tube to purify the DNA;

[0066] g: Repair the ends of the DNA fragments with PARP3 enzyme to have blunt and sticky ends;

[0067] h: Add sequencing adapters to both ends of the DNA fragments and make sure the concentration of the adapters matches the concentration of the DNA fragments;

[0068] i: Add a purification enzyme composition to the centrifuge tube to break down the PARP3 enzyme and unligated DNA sequencing adapters and improve the purity of the DNA;

[0069] S4: PCR amplification;

[0070] a: Design specific primers according to the sequence of the adapters;

[0071] b: Use high-fidelity DNA polymerase to PCR amplify the library;

[0072] c: Pour a purification enzyme composition into the centrifuge tube to remove impurities generated by PCR amplification and further improve the purity of the DNA;

[0073] S5: Sequencing reaction and data collection;

[0074] a: Pretreat the sequencing chip, including cleaning, coating, etc.

[0075] b: Load the amplified library onto the sequencing chip;

[0076] c: Perform sequencing reaction on the sequencer and capture the fluorescence signal at the end of the newly synthesized DNA strand;

[0077] d: Scan the sequencing chip with a laser to collect the fluorescence signal of each DNA cluster;

[0078] S6: Data analysis;

[0079] a: Perform quality control on the raw data obtained by sequencing, including removing low-quality sequences and removing adapter sequences;

[0080] b: align the processed sequences to the reference genome to determine the location of the sequences on the genome;

[0081] c: use bioinformatics tools to perform variant calling on the alignment results;

[0082] d: based on the results of variant calling, combined with the patient's clinical information and tumor characteristics, interpret the results and report.

[0083] In step S1-c, the centrifuge speed is 3000 rpm, and the centrifugation time is 10-15 min. Through the rotation of the low-speed centrifuge, the plasma and cell components can be separated.

[0084] In step S1-d, the standing time is 10-14 h, and the plasma sample is placed in a water bath at 55℃. By adding an appropriate amount of proteinase K to the plasma sample, the protein in the plasma can be digested. The digestion process is carried out at 55℃ water bath for 10-14 h to ensure that the protein is fully digested.

[0085] In the purification enzyme composition, trypsin and UDG enzyme are mixed in a weight ratio of 2:1.

[0086] In the mixer, trypsin and UDG enzyme are mixed at a stirring speed of 200 rpm, a stirring temperature of 37℃, and a stirring solution pH of 7.5. By stirring trypsin and UDG enzyme in this environment, trypsin and UDG enzyme can be mixed uniformly, and the activity of trypsin and UDG enzyme will not be affected.

[0087] In step S4-b, the number of PCR cycles is selected between 25-40 times, the denaturation temperature is 94-95℃ for 2-10 min, and the annealing temperature is 65℃ for 30-60 s. The number of PCR cycles refers to the number of times the three basic steps of denaturation, annealing (recombination), and extension are repeated in PCR reaction. Generally, the number of PCR cycles is selected between 25-40 times. The specific cycle number depends on the concentration of template DNA and the purpose of amplification. For example, if the concentration of template DNA is high, a smaller number of cycles (such as 25-30 times) can be selected; if the concentration of template DNA is low, the number of cycles needs to be increased to obtain sufficient amplification products (such as 30-40 times). However, it should be noted that too many cycles may increase the non-specific products.

[0088] In step S5-b, the concentration of the library needs to match the requirements of the sequencing chip. This can ensure the smooth loading process between the library and the sequencing chip, and reduce errors.

[0089] Wherein, in step S6-a, low-quality sequences and adapter sequences need to be removed. By removing low-quality sequences and adapter sequences, the error can be further reduced.

[0090] The data analyzed in step S6-c include single nucleotide polymorphisms (SNPs), insertions and deletions, and structural variations. The data of single nucleotide polymorphisms (SNPs), insertions and deletions, and structural variations are analyzed to obtain experimental data.

[0091] In this embodiment, a purified enzyme composition can be obtained by mixing trypsin and UDG enzymes at a weight ratio of 2:1. Furthermore, when the trypsin and UDG enzymes are mixed at a stirring speed of 200 rpm, a stirring temperature of 37° C., and a pH of 7.5, the activities of the trypsin and UDG enzymes are not affected, while the trypsin and UDG enzymes can be mixed together.

[0092] Furthermore, trypsin is a serine protease with broad substrate specificity. It can hydrolyze the peptide bonds formed by the carboxyl ends of lysine (Lys) and arginine (Arg) in peptide chains. PARP3 enzyme, as a specific enzyme, contains multiple amino acid residues in its structure, especially lysine (Lys) and arginine (Arg). These amino acid residues are connected by peptide bonds to form the polypeptide chain of PARP3 enzyme. This allows trypsin to match and hydrolyze the peptide bonds of PARP3 enzyme to achieve the purpose of decomposing PARP3 enzyme.

[0093] Furthermore, the purified enzyme composition can be used to decompose the PARP3 enzyme and sequencing adapters not ligated to DNA during the library construction step to improve DNA purity. It can also be used to decompose impurities generated during PCR amplification to further improve DNA purity. It is also worth noting that the purified enzyme composition can decompose the PARP3 enzyme, sequencing adapters not ligated to DNA, and impurities generated during PCR amplification, resulting in the production of amino acids such as aspartic acid, glutamine, and lysine, which do not affect the experimental structure of the next-generation sequencing.

[0094] The addition of a purification enzyme composition eliminates the magnetic bead purification required in steps S3-i and S4-c, replacing the magnetic bead purification process to achieve the desired removal effect and improve DNA purity. This purification enzyme composition, prepared using trypsin and UDG enzymes, is not only less expensive but also eliminates the problem of magnetic bead carryover, further improving measurement accuracy.

[0095] In the present invention, the PARP3 enzyme, sequencing adapters not connected to DNA, and impurities generated during PCR amplification can be decomposed by the purified enzyme composition. In this way, the magnetic bead purification step can be replaced by the purified enzyme composition in the library construction and PCR amplification steps. The purified enzyme composition prepared by trypsin and UDG enzyme is not only low in price, but also does not have the problem of residual magnetic bead particles, which can further improve the accuracy of the measurement results.

[0096] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for liquid biopsy of tumor DNA, comprising the following steps: S1: sample collection; a: Practical venous blood collection is used to collect blood from patients; b: Use EDTA anticoagulant tubes to preserve the collected blood; c) Place the EDTA anticoagulant tube into a centrifuge to separate the plasma and cell components; d: adding proteinase K to the plasma sample and allowing the plasma sample to stand for a period of time; e) extracting DNA from the plasma sample using a DNA extraction kit and placing the extracted DNA in a DNA storage tube; S2: DNA assessment; a: Use gel electrophoresis to detect DNA fragment size and integrity; b: DNA purity was measured using spectrophotometry; S3: Library construction; c) Pour Tn5 transposase, Tris-HCl buffer and DNA sample into a centrifuge tube and gently mix the reaction system; d: Incubate the reaction system at an appropriate temperature to allow the enzyme to fully act; e) When the DNA is broken to the desired length, ethylenediaminetetraacetic acid is added to the centrifuge tube to inhibit Tn5 transposase; f: Add ethanol precipitation and phenol to the centrifuge tube to purify the DNA; g: Use PARP3 enzyme to repair the ends of DNA fragments, making them have blunt and sticky ends; h: Add sequencing adapters to both ends of the DNA fragments and ensure that the concentration of the adapters matches the concentration of the DNA fragments; i: adding a purification enzyme composition into the centrifuge tube to decompose the PARP3 enzyme and the sequencing adapter that is not connected to the DNA, thereby improving the purity of the DNA; S4: PCR amplification; a: Design specific primers according to the sequence of the adapter; b: PCR amplification of the library using a high-fidelity DNA polymerase; c) Pour the purified enzyme composition into a centrifuge tube to remove impurities generated by PCR amplification and further improve the purity of the DNA; S5: sequencing reaction and data collection; a: Pre-processing the sequencing chip, including cleaning, coating, etc. b: Loading the amplified library onto the sequencing chip; c: Perform sequencing reaction on the sequencer and capture the fluorescent signal at the end of the newly synthesized DNA chain; d: Use laser scanning to scan the sequencing chip and collect the fluorescence signal of each DNA cluster; S6: Data analysis; a: Perform quality control on the raw data obtained from sequencing, including removing low-quality sequences and adapter sequences; b: Align the processed sequence with the reference genome to determine the position of the sequence on the genome; c: Use bioinformatics tools to compare the results and perform variant detection; d: Based on the results of variant detection, combined with the patient's clinical information and tumor characteristics, the results are interpreted and reported.

2. A tumor DNA liquid biopsy detection method according to claim 1, characterized in that: The centrifuge speed in step S1-c is 3000 rpm, and the centrifugation time is 10-15 min.

3. The method for liquid biopsy of tumor DNA according to claim 1, characterized in that: The standing time in step S1-d is 10-14 hours, and the plasma sample is placed in a water bath at 55°C.

4. A tumor DNA liquid biopsy detection method according to claim 1, characterized in that: The purified enzyme composition is prepared by mixing trypsin and UDG enzyme, wherein the weight ratio of trypsin to UDG enzyme is 2:

1.

5. A tumor DNA liquid biopsy detection method according to claim 4, characterized in that: The trypsin and UDG enzyme were mixed in a stirrer at a stirring speed of 200 rpm, a stirring temperature of 37° C., and a pH of the stirred solution of 7.

5.

6. The method for liquid biopsy of tumor DNA according to claim 1, characterized in that: The number of PCR cycles in step S4-b is selected to be between 25 and 40 times, the denaturation temperature is 94-95°C and lasts for 2-10 minutes, and the annealing temperature is 65°C and lasts for 30-60 seconds.

7. The method for liquid biopsy of tumor DNA according to claim 1, characterized in that: In step S5-b, it is necessary to ensure that the concentration of the library matches the requirements of the sequencing chip.

8. The method for liquid biopsy of tumor DNA according to claim 1, characterized in that: In step S6-a, low-quality sequences and adapter sequences need to be removed.

9. The method for liquid biopsy of tumor DNA according to claim 1, characterized in that: The data analyzed in step S6-c include single nucleotide polymorphisms (SNPs), insertions and deletions, and structural variations.