WO2016109939A1 - 一种富集循环肿瘤dna的方法和试剂 - Google Patents

一种富集循环肿瘤dna的方法和试剂 Download PDF

Info

Publication number
WO2016109939A1
WO2016109939A1 PCT/CN2015/070188 CN2015070188W WO2016109939A1 WO 2016109939 A1 WO2016109939 A1 WO 2016109939A1 CN 2015070188 W CN2015070188 W CN 2015070188W WO 2016109939 A1 WO2016109939 A1 WO 2016109939A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
circulating tumor
tumor dna
aqueous phase
sequence
Prior art date
Application number
PCT/CN2015/070188
Other languages
English (en)
French (fr)
Inventor
许明炎
张晓妮
Original Assignee
深圳市海普洛斯生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市海普洛斯生物科技有限公司 filed Critical 深圳市海普洛斯生物科技有限公司
Priority to CN201580006707.6A priority Critical patent/CN106103742B/zh
Priority to PCT/CN2015/070188 priority patent/WO2016109939A1/zh
Priority to US15/127,736 priority patent/US9944993B2/en
Publication of WO2016109939A1 publication Critical patent/WO2016109939A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the field of PCR (Polymerase Chain Reaction) ultra-micro parallel amplification technology, in particular to a method and a reagent for enriching circulating tumor DNA.
  • PCR Polymerase Chain Reaction
  • genomic DNA is mainly derived from tissue cells, including: extraction of cells after rupture of living tissue cells; in vitro PCR amplification of tissue cell paraffin samples. For early patients, how to diagnose cancer from DNA level, or even prevent it, there are still technical barriers.
  • the five-year survival rate of liver cancer and lung cancer with poor prognosis is also higher than that of China, reaching 15%-20% (related The data comes from Lancet). It can be seen that to improve the five-year survival rate of cancer patients in China, it is necessary to start from the "three early mornings", early detection, early diagnosis and early treatment.
  • Imaging includes: X-ray (chest fluoroscopy, chest X-ray, low-dose spiral CT), magnetic resonance (MIR), radioactive material (nuclear bone scan), PET-CT.
  • X-ray as a routine screening method, mainly to determine the size and location of the tumor, to be diagnosed also need to be combined with the detection of tumor markers; MIR to detect whether the tumor spread to the brain or spinal cord, nuclear orbital scan is to detect whether the tumor is Diffusion to bones, these two detection techniques are mainly used for the diagnosis of tumor after stage III; PET (Positron Emission Computed Tomography), full name positron emission tomography, is the only anatomical way to function, metabolism and receptor The imaging technique is non-invasive. PET-CT is a combination of PET technology and CT to screen the location and spread of tumors. It is clinically used for diagnosis and One of the best ways to guide the treatment of cancer.
  • tumor markers need to be evaluated, and tumors can be identified or diagnosed based on their biochemical or immunological properties.
  • a tumor marker is a substance produced and released by a tumor cell, and is often present in a tumor cell or in a host body fluid in the form of a metabolite such as an antigen, an enzyme, or a hormone.
  • Tumor markers for clinical detection include alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen family (CA125, CA15-3, etc.) and the like.
  • the malignant tumors detected by conventional imaging studies of the above clinical applications generally have a diameter of more than 1 cm, the number of tumor cells reaches 10 9 and the weight exceeds 1 g.
  • PET-CT has high sensitivity and can detect tumors above 0.5 cm (H. Li et al. 2013; Bu Zhaode, Xue Zhongyu et al., 2000). That is to say, most of the tumor patients detected by imaging are in the middle and late stage, and have missed the best period of cancer treatment.
  • the early stage of cancer even if a tumor marker is detected, the specific location of the tumor tissue cannot be detected by imaging, and the tumor cannot be finally diagnosed, and the early treatment and treatment can only be allowed to deteriorate.
  • Tumor drug resistance and recurrence after healing are one of the most important causes of death in cancer patients. Solving these two problems has become a hot research topic in the world of cancer diseases. In the course of cancer treatment, current clinical applications lack an effective means of drug evaluation. Taking lung cancer as an example, gefitinib (Iressa) and erlotinib (Troquet) are treatments for non-small cell lung cancer (NSCLC), and clinical data indicate that not all patients with non-small cell lung cancer are suitable. Use such drugs. Further studies have found that patients with EGFR (epidermal growth factor receptor) mutations receive more than 40% more response to Iressa than to non-selective patients (Tony S. Mok, MD et al.
  • EGFR epidermatitis
  • Gemcitabine is a difluoronucleoside anti-metabolite anticancer drug that destroys cell replication and is suitable for the treatment of advanced non-small cell lung cancer. Studies have shown that patients with low expression of RRM1 (RRM1 gene located in the short arm of chromosome 1 encoding the ribonucleotide reductase M1 subunit) received gemcitabine for more than 30% disease control (Lee et al. 2010).
  • the prognosis of clinical treatment of cancer there is also a blind period of detection, for example: many cancer patients surgically remove the tumor tissue to prevent its continued spread and life-threatening, postoperative regular biopsy for prognosis evaluation. Because of the large radiation and damage to the human body, the cancer patients are reviewed on a yearly basis, which greatly affects the timeliness of prognosis evaluation. It also reflects the urgent need for a simple and no-diagnosis in the field of cancer diagnosis. Traumatic testing technology. All in all, the current clinical medicine screening, diagnosis, treatment and prognosis of early cancer is a major breakthrough in cancer treatment and the key to improving the five-year survival rate of cancer. Whether in medical research or in clinical applications, there are major technical challenges in this area.
  • Circulating tumor DNA is a tumor cell's somatic DNA that is shed or released into the circulatory system after apoptosis, and can be characterized, quantified, and traced.
  • ctDNA Circulating tumor DNA
  • the invention provides a method and a reagent for enriching circulating tumor DNA, which can effectively capture ctDNA in peripheral blood plasma, and perform single molecule high fidelity ultra-micro parallel amplification to provide sufficient amount of ctDNA for subsequent sequencing. Detection use.
  • the invention provides a method of enriching circulating tumor DNA, comprising the steps of:
  • Parallel amplification of single molecule mixing the aqueous phase and the oil phase, oscillating into an emulsion PCR reaction system, and performing emulsion PCR amplification, wherein the aqueous phase contains peripheral blood plasma DNA, forward and reverse primers, and dNTPs as template DNA.
  • a PCR buffer and a DNA polymerase wherein the two ends of the peripheral blood plasma DNA are linked to a linker sequence, and the forward and reverse primers are complementary to the linker sequences at both ends;
  • Separating the aqueous phase and the oil phase after the emulsion is amplified by PCR, the aqueous phase and the oil phase are separated to obtain a PCR amplification product of the aqueous phase;
  • Capture of circulating tumor DNA The circulating tumor DNA in the PCR amplification product of the aqueous phase is captured using a probe sequence that specifically binds to circulating tumor DNA.
  • the aqueous phase of the emulsion PCR reaction system further contains dUTP;
  • the molar ratio of the dUTP to the dNTPs is from 1:1000 to 1:10, more preferably 1:100.
  • the linker sequence is a sequencing linker sequence used in a sequencing device
  • the sequencing device is an Illumina sequencer
  • the forward and reverse primer sequences corresponding to the sequencing linker sequence are 5'-TCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 1) and 5'-TGAACCTGAACCGCTCTTCCGATCT-3' (SEQ ID NO: 2);
  • the sequencing device is a Life Tech sequencer
  • the forward and reverse primer sequences corresponding to the sequencing linker sequence are 5'-CCATCTCATCCCTGCGTGTCTCCGA-3' (SEQ ID NO: 3) and 5'-CCGCTTTCCTCTCTATGGGCAGTCG-3', respectively. SEQ ID NO: 4).
  • the volume ratio of the aqueous phase to the oil phase is from 1:10 to 1:1, preferably from 1:5 to 1:2.
  • the DNA polymerase is a high-fidelity DNA polymerase, preferably a high-fidelity Klenow Fragment (DNA polymerase I, DNA Polymerase I), a KAPAHiFi family high-fidelity DNA polymerase, and a Phusion family high-fidelity DNA polymerization. Enzyme or Q5 family high fidelity DNA polymerase.
  • the total amount of peripheral blood plasma DNA in the aqueous phase is 1-100 ng
  • the final concentration of the positive and negative primers is 0.1-1 ⁇ M
  • the final concentration of dNTPs is 0.5-2 mM
  • the final concentration of the PCR buffer is 1 time.
  • the final concentration of DNA polymerase is 0.1-1 U;
  • the total amount of peripheral blood plasma DNA in the aqueous phase is 1-10 ng
  • the final concentration of the positive and negative primers is 0.5 ⁇ M
  • the final concentration of dNTPs is 1 mM
  • the final concentration of the PCR buffer is 1 time
  • the final concentration of the DNA polymerase is 0.25. U;
  • the procedure for PCR amplification of the emulsion is 93-95 ° C for 1-3 min; 93-95 ° C for 5-20 s, 61-65 ° C 10-20s, 71-73 ° C 5-20s, 40-60 cycles; 71-73 ° C 4-10min;
  • the procedure for emulsion PCR amplification is 94 ° C for 2 min; 94 ° C for 10 s, 63 ° C for 15 s, 72 ° C for 10 s, 50 cycles; 72 ° C for 5 min.
  • the probe sequence for specifically binding to circulating tumor DNA used in the capture circulating tumor DNA carries biotin modification; after the probe sequence specifically binds to the circulating tumor DNA, passes through the chain
  • the mycelium magnetic beads specifically bind to biotin to capture the circulating tumor DNA; it should be noted that the biotin modification can be at any position of the probe sequence, such as the 5' end, the 3' end or the middle of the probe sequence. Any base position.
  • the linker sequence is blocked using a blocking sequence, wherein the blocking sequence specifically binds to a linker sequence at both ends of the peripheral blood plasma DNA.
  • the capture of circulating tumor DNA is specifically achieved by PCR amplification using a probe sequence that specifically binds circulating tumor DNA as a primer, wherein the probe sequence as a primer is specifically bound.
  • the sequence of circulating tumor DNA is specifically achieved by PCR amplification using a probe sequence that specifically binds circulating tumor DNA as a primer, wherein the probe sequence as a primer is specifically bound.
  • the invention provides an agent enriched for circulating tumor DNA, the reagent comprising the following components:
  • the emulsion PCR amplification component comprises an aqueous phase comprising a forward and reverse primer, dNTPs, a PCR buffer and a DNA polymerase, wherein the positive and negative primers are respectively linked to peripheral blood plasma DNA. Complementary pairing of the adaptor sequences at the ends;
  • a circulating tumor DNA component comprising a probe sequence that specifically binds to circulating tumor DNA, for capturing circulating tumor DNA in a PCR amplification product of the aqueous phase;
  • the aqueous phase of the emulsion PCR amplification component further comprises dUTP;
  • the molar ratio of the dUTP to the dNTPs is from 1:1000 to 1:10, more preferably 1:100:
  • the DNA polymerase is a high-fidelity DNA polymerase, preferably a high-fidelity Klenow Fragment (DNA Polymerase I), a KAPA HiFi family high-fidelity DNA polymerase, a Phusion family high-fidelity DNA polymerase or a Q5 family high-fidelity DNA. Polymerase.
  • the probe sequence carries a biotin modification
  • the reagent further comprises a streptavidin magnetic bead
  • the probe sequence specifically binds to the circulating tumor DNA, passes through the chain
  • the mycelium magnetic beads specifically bind to biotin to capture the circulating tumor DNA
  • the reagent further comprises a blocking sequence that specifically binds to peripheral blood blood a linker sequence at both ends of the plasma DNA for blocking the linker sequence during capture of the circulating tumor DNA using the probe sequence;
  • the probe sequence is a sequence that specifically binds to circulating tumor DNA, and is used for PCR amplification to obtain enriched circulating tumor DNA.
  • the method of the invention combines emulsion PCR with probe capture technology to achieve single molecule high fidelity ultra-micro parallel amplification and efficient capture of ctDNA in peripheral blood plasma, providing a sufficient amount of ctDNA for subsequent sequencing detection.
  • the method of the invention does not require tissue cell biopsy sampling, as long as a small amount of peripheral blood plasma can enrich ctDNA in large quantities for subsequent sequencing detection, thereby achieving non-invasive detection. It not only saves the detection cost, improves the diagnostic accuracy, but also breaks the detection limit of cancer, from the traditional middle and late detection, advanced to early diagnosis, and the method of the invention is simple and convenient to operate.
  • the method for enriching circulating tumor DNA of the present invention itself cannot determine the cancer condition of the patient, because the method itself is only for enriching circulating tumor DNA in peripheral blood plasma, and is sufficient for subsequent sequencing analysis to carry information.
  • the sample material therefore, the method of enriching circulating tumor DNA itself cannot be used as a diagnostic method.
  • FIG. 1 is a schematic diagram showing the principle and process of emulsion PCR in the present invention
  • FIG. 2 is a schematic diagram showing the principle and process of biotin-oligonucleotide capture ctDNA in the present invention
  • Example 3 is a result of gel electrophoresis detection of ctDNA captured in Example 1 of the present invention.
  • FIG. 4 is a schematic diagram showing the principle and process of PCR capture of ctDNA by primers in the present invention
  • Example 5 is a result of gel electrophoresis detection of ctDNA captured in Example 2 of the present invention.
  • Fig. 6 is a result of gel electrophoresis detection of ctDNA captured in Example 3 of the present invention.
  • the invention mainly solves the following two problems:
  • Capture problem of ctDNA For the above parallel-amplified DNA containing ctDNA and nDNA, it is necessary to capture the cancer gene portion from the whole genome to obtain ctDNA, and the method of the present invention successfully realizes ctDNA capture by capturing the probe sequence of ctDNA. set.
  • the method of the present invention is capable of efficiently capturing ctDNA, performing single-molecule high-fidelity amplification, and providing a sufficient amount of ctDNA for subsequent detection.
  • the method of the present invention mainly comprises two parts, and solutions are respectively proposed for the above two problems.
  • the first part is the solution to the first problem: single-molecule parallel amplification of ctDNA and nDNA using emulsion PCR (ePCR).
  • ePCR emulsion PCR
  • the second part is the solution to the second problem: hybridization of the cancer gene with biotin-oligo, followed by streptavidin magnetic beads, capture and isolate Cancer gene; or design primers, PCR amplification of ctDNA, capture and isolate cancer genes.
  • emulsion PCR is a PCR amplification using micro droplets in a water-in-oil emulsion system as a reactor.
  • the ePCR system includes a reaction system of an oil phase and an aqueous phase.
  • the oil phase may be used as a carrier, and the oil phase used in the present invention may be not limited, and an oil phase system shown in Table 1 may preferably be used.
  • the reaction system in the aqueous phase includes: DNA template (DNA template), dNTPs (including dATP, dTTP, dCTP, dGTP), PCR buffer, DNA polymerase (DNA polymerase) and double distilled water (ddH 2 O).
  • DNA template selects DNA in peripheral blood plasma, which contains double-stranded ctDNA and nDNA.
  • the DNA duplex Prior to ePCR, the DNA duplex is subjected to a three-step modification, the first step of repairing the double-stranded DNA ends; the second step is to add "A" to the 3' end of the DNA fragment; and the third step, at the end of the DNA Specific adapters are used for subsequent sequencing, and the selection of the adapters is combined with specific sequencing equipment (common sequencing equipment such as illumina, Life Tech, etc.).
  • specific sequencing equipment common sequencing equipment such as illumina, Life Tech, etc.
  • the sequence of the linker primers is as follows:
  • Forward primer (5'-3'): TCCCTACACCGGCCTCTTCCGATCT (SEQ ID NO: 1); reverse primer (5'-3'): TGAACCTGAACCGCTCTTCCGATCT (SEQ ID NO: 2).
  • Ion_A (Red, 5'-3'): CCATCTCATCCCTGCGTGTCTCCGA (SEQ ID NO: 3); Ion_P1 (Blue, 5'-3'): CCGCTTTCCTCTCTATGGGCAGTCG (SEQ ID NO: 4).
  • dNTPs aqueous phase reaction system
  • dUTP aqueous phase reaction system
  • buffer aqueous phase reaction system
  • DNA polymerase aqueous phase reaction system
  • the other components of the aqueous phase reaction system dNTPs, dUTP, buffer, DNA polymerase can be configured as Master Mix in advance, see Table 2, and placed at -20 °C for storage after use. .
  • the aqueous phase/oil phase can be selected in different ratios, such as: 75 ⁇ L/400 ⁇ L; 240 ⁇ L/960 ⁇ L; 200 ⁇ L/400 ⁇ L; 300 ⁇ L/400 ⁇ L.
  • the ePCR system and cycle parameters in the aqueous phase are shown in Tables 3 and 4.
  • the aqueous and oil phases were sequentially added to a 1.5 mL non-stick reaction tube. After the two phases were mixed, the reaction tube containing the reaction system was placed in a tissue disrupter (Qiagen Tissue Lyser II) for 90 seconds, and the parameter was set to 13 Hz. The reaction tube was taken out, and the oil-water system was dispensed into a PCR reaction tube, and the PCR reaction was carried out according to the setting parameters of Table 4.
  • tissue disrupter Qiagen Tissue Lyser II
  • Method 1 remove the oil phase in the upper part of the reaction tube, add 400 ⁇ L of paraffin oil to the reaction tube, vortex or pipette the solution in the tube repeatedly until the emulsion of the aqueous phase is broken. The aqueous and oil phases are thoroughly mixed. Transfer the mixed liquid from the PCR reaction tube to a new non-stick wall reaction tube, elute the PCR reaction tube with 400 ⁇ L of silicon oil, and add the eluent to the above aqueous/oil phase mixture, and vortex well. Centrifuge at 15,000 rpm for 2 min to remove the upper oil phase.
  • the silicone oil was eluted three times, 500 ⁇ L of silicone oil was added, vortexed thoroughly, centrifuged at 15,000 rpm for 1 min, and the upper oil phase was discarded, and the operation was repeated twice.
  • the PCR reaction product separated into the aqueous phase.
  • Method 2 Collect the aqueous phase and the oil phase in the reaction tube, centrifuge at 9000 g for 5 min, and remove the oil phase. At this time, the aqueous phase still precipitates as a pellet in the bottom of the reaction tube, and 400 ⁇ L (two volumes) of saturated diethyl ether is added, and the mixture is fully vortexed.
  • Method 3 Collect the aqueous phase and the oil phase in the reaction tube, discard the upper oil phase by centrifugation at 15000 rpm for 10 min, add 400 ⁇ L (two volumes) of phenol/chloroform, mix the mixture thoroughly, centrifuge at 15000 rpm for 2 min, and remove the upper phenol. /Trichloromethane; phenol/trichloromethane is washed once to obtain a PCR reaction product of the aqueous phase.
  • the obtained DNA product is a single-stranded DNA containing a linker at both ends, in order to avoid capturing ctDNA.
  • the linker at the end of the DNA is complementary to introduce nDNA, and the obtained ePCR product is added to the oligonucleotide sequence complementary to the linker in the subsequent experiment, that is, the finally obtained DNA single strand, which is in the form of a double-linker at the end.
  • the system has DNA polymerase and the corresponding buffer, in the next experiment, consider whether the enzyme needs to be inactivated (generally 65-70 ° C, 10 min, the specific temperature depends on the enzyme used).
  • Part II The first part of the single-stranded DNA is obtained. Here, ctDNA is captured. There are two options, which are listed below.
  • Protocol 1 Please refer to Figure 2, mainly to capture single-stranded ctDNA by magnetic bead-specific biotin-labeled oligonucleotide (biotin-oligo).
  • biotin-oligo magnetic bead-specific biotin-labeled oligonucleotide
  • Biotinylated oligonucleotides comprising two parts: a gene complementary pairing moiety and a biotinylated moiety.
  • Biotin can be specifically adsorbed on the surface of streptavidin magnetic beads, and the ctDNA can be isolated by separating the magnetic beads, washing away the other components and eluting; the complementary pairing portion of the gene is basified with ctDNA during the process of capturing ctDNA. Complementary pairing binding.
  • the length of the complementary pair of genes can be adjusted according to specific conditions, generally about 50 bp.
  • the designed biotin-labeled oligonucleotide was added, placed at 98 ° C for 5 min, shaken at 60 ° C for 2 hours, and then cooled to room temperature. Add the washed streptavidin magnetic beads and shake at room temperature for 30 min. The magnetic beads must be below the liquid level to ensure that the magnetic beads are fully bound to the biotinylated ctDNA-Biotin-oligo complex. Next, the magnetic beads are eluted.
  • the magnetic beads are placed on the wall of the tube, remove the liquid, and repeat the operation twice until the liquid is cleaned; add 300 ⁇ L of 1 ⁇ B&w buffer to wash, quickly vortex, and place on the magnetic stand. Place for 2 min, the magnetic beads are placed on the tube wall, remove the liquid, and repeat this operation; add 300 ⁇ L of 1 ⁇ TE buffer to wash, vortex rapidly, place on the magnetic stand for 2 min, and the magnetic beads are placed on the tube wall. Remove the liquid and repeat this operation. Finally, add 300 ⁇ L of 0.125 M NaOH solution, vortex for 1 sec, place at room temperature for 10 min, then place it on the magnetic stand for 2 min, and place the magnetic beads on the tube wall. The clear liquid is the captured ctDNA.
  • the magnetic beads can be reused after eluting three times with 1 ⁇ TE buffer.
  • the specific operation is as follows: the first elution, adding 300 ⁇ L of 1 ⁇ TE buffer, vortexing and then rapidly centrifuging, placed in a magnetic stand for 30 sec, the magnetic beads are closely attached to the tube wall, and the supernatant is removed; the second elution, Add 300 ⁇ L of 1 ⁇ TE buffer (containing 0.01% Trition), vortex, centrifuge rapidly, place on a magnetic stand for 30 sec, the magnetic beads are placed on the tube wall to remove the supernatant; the third elution, add 300 ⁇ L 1 ⁇ TE Buffer (containing 0.01% Trition), vortexed quickly and centrifuged, placed in magnetic The force frame was placed for 30 sec, the magnetic beads were placed close to the tube wall, and the supernatant was removed. Finally, the magnetic beads were suspended in 50 ⁇ L of 1 ⁇ TE buffer (containing 0.01% Trition).
  • Blood samples from patients with lung cancer were stored in a procoagulant blood collection tube. 1 mL of the supernatant was taken, placed in a 1.5 mL EP tube, centrifuged at 13,000 rpm for 1 min, the pellet was discarded, and the supernatant was used.
  • step 6 CR2 was placed in a centrifuge, centrifuged at 12000 rpm for 2 min, and allowed to stand at room temperature for 5 min; 8, 40 ⁇ L of TB buffer was used to elute CR2, left at room temperature for 5 min, and then centrifuged at 12000 rpm for 2 min to allow sufficient elution. This step can be repeated once.
  • the extracted serum plasma DNA concentration was 6.74 ng/ ⁇ L.
  • the extracted serum plasma DNA was subjected to ePCR, and the aqueous phase/oil ratio was 200 ⁇ L/400 ⁇ L.
  • the oil phase was prepared according to Formulation 3 of Table 1, 700 ⁇ L of emulsifier ABIL WE90, 200 ⁇ L of paraffin oil, and 730 ⁇ L of diethylhexyl carbonate in 1 mL of the oil phase.
  • the components dNTPs, dUTP, buffer, and DNA polymerase in the aqueous phase system were configured as Master Mix according to Table 2, wherein 20 ⁇ L of 10X buffer, 5 ⁇ L of phi29 DNA polymerase (10 U/ ⁇ L), and 20 ⁇ L of dNTPs (10 mM).
  • the DNA template was added in an amount of 1 ⁇ L, so that the aqueous phase was 200 ⁇ L in total.
  • PCR parameters set three steps, first step, pre-denaturation, 94 ° C, 2 min; second cycle operation, denaturation, 94 ° C, 10 sec, primer annealing, 63 ° C, 15 sec, extension 72 ° C, 10 sec, a total of 35 Cycle; third step, final extension, 72 ° C, 5 min.
  • the obtained ePCR reaction product was stored at 4 ° C for use.
  • the product of the ePCR is subjected to post-treatment to separate the aqueous phase and the oil phase.
  • the DNA product amplified by the EPCR contains the DNA polymerase. Therefore, before the next experiment, the DNA polymerase is inactivated, and the reaction tube is incubated at 65 ° C for 10 min.
  • the DNA product obtained from ePCR captures ctDNA and designs a biotin-labeled oligonucleotide (Biotin-oligo).
  • Biotin-oligo Biotin-oligo
  • the 17th exon (exon 17th) sequence of EGFR to be captured is as follows (5'-3'):
  • GGCCCCCACCA-3' (SEQ ID NO: 6), wherein the 5'-end portion carries a biotin label.
  • a 5'-terminal portion carrying a biotin-labeled oligonucleotide was added to capture ctDNA.
  • the magnetic beads must be below the liquid level to ensure that the magnetic beads are fully bound to the biotinylated ctDNA-Biotin-oligo complex.
  • Adsorb the magnetic beads Place the reaction tube on the magnetic stand for 2 min, the magnetic beads are placed on the tube wall, the liquid is removed, and the operation is repeated twice until the liquid is removed. 4. Elute the magnetic beads. Add 300 ⁇ L of 1 ⁇ B&w buffer to wash, vortex rapidly, place on a magnetic stand for 2 min, place the magnetic beads on the tube wall, remove the liquid, repeat this operation again; add 300 ⁇ L of 1 ⁇ TE buffer to wash, vortex, Place it on the magnetic stand for 2 min, the magnetic beads are placed on the tube wall, remove the liquid, and repeat this operation. 4.
  • the captured DNA product was 142 bp in size. Take 5 ⁇ L of the captured DNA solution, add 1 ⁇ L of 6X loading buffer, mix thoroughly, and perform electrophoresis.
  • the DNA marker was 100 bp plus (GENSTAR) and loaded with 1 ⁇ L.
  • the agarose gel was set to a concentration of 2%, and 2 g of agarose was added to 100 mL of 1X TAE buffer. The mixture was heated in a microwave for 2 min until the agarose was completely dissolved. The solution was cooled to 50-60 ° C, and 5 ⁇ L of nucleic acid dye was added (Gold View). ), pour the glue.
  • PCR primers were designed to include two parts: a gene complementary pairing portion and an adapter portion.
  • the PCR primer does not require terminal modification, the gene complementary pairing portion is located at the 3' end, and base pairing with single-stranded ctDNA occurs during PCR amplification; the linker portion is located at the 5' end, and plays a role in subsequent gene sequencing.
  • the length of the complementary pair of genes can be adjusted according to the specific conditions, generally about 25 bp, not less than 20 bp; in the design of the linker, combined with the specific sequencing equipment selection sequence, the length is generally not less than 20 bp.
  • the PCR synthetase generally uses high-purity Klenow Fragment (DNA Polymerase I), KAPAHiFi family, high-fidelity Phusion family, and high-fidelity Q5 family DNA polymerase. Therefore, when designing primers, the primer annealing temperature should be consistent with the activity temperature of the selected synthetase.
  • the DNA product amplified by the ePCR contains the DNA polymerase. Therefore, before the next experiment, the DNA polymerase is inactivated, and the reaction tube is incubated at 65 ° C for 10 minutes.
  • Reverse primer (5'-3') CTCCCTCTCCTGCAGCAGCCTC (SEQ ID NO: 8).
  • Reverse primer (5'-3') TGAACCTGAACCGCTCTTCCGATCT (SEQ ID NO: 2).
  • Tm represents the annealing temperature (salt concentration is 50 mM):
  • TCCCTACACGACGCTCTTCCGATCTGCCTAAGATCCCGTCCATCGCCA SEQ ID NO: 9, Tm: 69.7 ° C
  • the DNA product obtained by ePCR was 200 ⁇ L, and the concentration was 50 ng/ ⁇ L, and 1 ⁇ L was diluted 10-fold to obtain a DNA solution having a concentration of 5 ng/ ⁇ L. PCR amplification was carried out using this concentration of DNA solution as a template.
  • the PCR amplification system and cycle parameters are shown in Tables 4 and 5 below.
  • the synthetase is KAPA2G Robust HotStart.
  • PCR product the size of 192 bp (adapter with primers at both ends), take 5 ⁇ L of PCR reaction product, add 1 ⁇ L of 6X loading buffer (loading buffer), mix well, sample for electrophoresis detection, DNA marker select 100bp plus (GENSTAR) , load 1 ⁇ L.
  • concentration of the agarose gel was 2%, and the configuration was the same as in Example 1.
  • the results of electrophoresis detection are shown in Figure 5.
  • the captured DNA solution was loaded into the ninth lane, and according to the electropherogram analysis, the size of the target band was consistent, that is, the method successfully captured ctDNA.
  • the DNA obtained by this PCR was the 17th exon of EGFR, and the PCR product was sequenced for one generation.
  • the system was expanded to perform nucleic acid gel electrophoresis, and the concentration of the agarose gel was 2%, and the gel was recovered. The remaining 95 ⁇ L of the PCR reaction product, plus 19 ⁇ L of 6X loading buffer, was thoroughly mixed and spotted for electrophoresis.
  • the DNA marker was 100 bp plus (GENSTAR) and loaded with 6 ⁇ L.
  • the agarose gel is prepared as above.
  • the above PCR product is subjected to gel recovery (OMEGA gel recovery kit), and the specific operation is as follows: 1.
  • the nucleic acid gel containing the target band EGFR gene exon17 is cut under ultraviolet light, and the weight is 0.50000 g (the blank EP tube weight is 1.04100 g, and the cutting is added. Gel weight 1.54100g);
  • the recovered product (30 ng/ ⁇ L, ⁇ 10 ⁇ L) was sent to a biotech for one-generation sequencing, and the forward primer was sequenced.
  • the PCR product sequence was aligned with the exton17 sequence of the EGFR gene.
  • the peak of the 1-50 bp DNA base appeared to be encapsulated.
  • the serum-extracted DNA contains nDNA and ctDNA, and the ratio of the two is about 1000:1, so a peak appears, indicating that the part of the DNA contains nDNA and ctDNA, and the ctDNA sequence may be mutated.
  • the DNA product amplified by the ePCR contains the DNA polymerase. Therefore, before the next experiment, the DNA polymerase is inactivated, and the reaction tube is incubated at 65 ° C for 10 minutes.
  • the resulting DNA product was amplified by EPCR, and ctDNA was captured by primer PCR.
  • Designing PCR primers, the 17th exon (exon 17th) sequence of EGFR to be captured is as follows (5'-3'):
  • Reverse primer (5'-3') CTCCCTCTCCTGCAGCAGCCTC (SEQ ID NO: 8).
  • Ion_A (Red, 5'-3'): CCATCTCATCCCTGCGTGTCTCCGA (SEQ ID NO: 3);
  • Ion_P1 (Blue, 5'-3'): CCGCTTTCCTCTCTATGGGCAGTCG (SEQ ID NO: 4).
  • 200 ⁇ L of the DNA product obtained by EPCR was measured at a concentration of 50 ng/ ⁇ L, and diluted 10 times with 1 ⁇ L to obtain a DNA solution having a concentration of 5 ng/ ⁇ L.
  • PCR amplification was carried out using this concentration of DNA solution as a template.
  • the PCR amplification system and cycle parameters are shown in Tables 6 and 7 below.
  • the synthetase is Phusion DNA Polymerase (Phusion DNA Polvmerase).
  • PCR product size 192 bp (adapter with primers at both ends), take 5 ⁇ L of PCR reaction product, add 1 ⁇ L of 6X loading buffer (loading buffer), mix thoroughly, and perform electrophoresis detection by spotting.
  • DNA marker selects 100bp plus (GENSTAR) ), load 1 ⁇ L.
  • the agarose gel had a concentration of 2% and was prepared in the same manner as in Example 1.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pathology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明公开了一种富集循环肿瘤DNA的方法和试剂，所述方法包括如下步骤：将水相和油相混合、振荡配成乳液PCR反应体系并进行乳液PCR扩增，其中水相中含有作为模板DNA的外周血液血浆DNA、正反向引物、dNTPs、PCR缓冲液和DNA聚合酶，外周血液血浆DNA的两端连有接头序列，正反向引物分别与两端的接头序列互补配对；乳液PCR扩增后，分离水相和油相，得到水相的PCR扩增产物；使用特异性结合循环肿瘤DNA的探针序列捕获水相的PCR扩增产物中的循环肿瘤DNA。本发明的方法能够有效地捕获外周血液血浆中的ctDNA，并且进行单分子高保真超微量平行扩增，提供了足够量ctDNA以进行后续的测序检测使用。

Description

一种富集循环肿瘤DNA的方法和试剂 技术领域
本发明涉及PCR(Polymerase Chain Reaction，聚合酶链式反应)超微量平行扩增技术领域，尤其涉及一种富集循环肿瘤DNA的方法和试剂。
背景技术
2003年以来，随着人类基因组计划的开展，一代测序(Sanger)和二代测序(NGS)技术以其灵敏度和准确性逐步占据分子诊断的市场。分子水平的基因组学也成为了21世纪生命科学领域的研究热点。基因测序技术的趋于临床应用化，各种癌症相关基因的发现，癌症基因组计划的展开，为癌症的早期诊断、个体化治疗，以及预后带来了新的曙光。目前，基因组DNA的获得主要来源于组织细胞，包括：活体组织细胞破裂后提取；组织细胞石蜡样本体外PCR扩增。而对于早期的患者，如何从DNA水平诊断癌症的发生，甚至预防，这仍然存在技术壁垒。
恶性肿瘤的生存检测数据是反映一个地区肿瘤负担以及评价医疗资源和防治水平的重要信息。我国全国肿瘤登记中心的2003-2005年癌症生存率分析表明，在我国癌症5年相对生存率仅为30.9％。主要癌症的生存率分别为：肺癌16.1％，胃癌27.4％，肝癌10.1％，食管癌20.9％，结直肠癌47.2％，乳腺癌73％。其中，城乡癌症生存率差异明显。而在发达国家，结直肠癌和乳腺癌的五年生存率，分别达到60％和85％，预后较差的肝癌和肺癌的五年生存率也高于我国，达到15％-20％(相关数据来自Lancet)。可见，要提高我国癌症患者的五年生存率，需从“三早”做起，早发现，早诊断，早治疗。
目前，临床应用的癌症诊断和评估手段主要是影像学定位和肿瘤标记物定性相结合。影像学包括：X射线(胸部透视、X线胸片、低剂量螺旋CT)、磁共振(MIR)、放射性物质(核骨素扫描)、PET-CT。X射线作为一种常规的筛查方法，主要确定肿瘤的大小和位置，要进行确诊还需结合肿瘤标志物的检测；MIR检测肿瘤是否扩散到脑或者脊髓，核骨素扫描则是检测肿瘤是否扩散到骨头，这两种检测技术主要用于肿瘤III期之后的诊断；PET(Positron Emission Computed Tomography)，全称正电子发射计算机断层扫描，是目前唯一的用解剖形态方式进行功能、代谢和受体显像的技术，具有无创伤性的特点，PET-CT是PET技术和CT相结合，筛查肿瘤的位置和扩散情况，是临床上用以诊断和 指导治疗肿瘤最佳手段之一。但是，这种诊断方法费用较高，且不属于医保范畴，很多患者因无法支付昂贵的检查费而不得不放弃使用。影像学的检测主要用于恶性肿瘤的定位，要定性癌症的类别和分期，还需要对肿瘤标志物进行评估，根据其生化或免疫特性可以识别或诊断肿瘤。肿瘤标记物是由肿瘤细胞产生和释放的某种物质，常以抗原、酶、激素等代谢产物的形式存在于肿瘤细胞内或宿主体液中。用于临床检测的肿瘤标记物有：甲胎蛋白(AFP)、癌胚抗原(CEA)、糖类抗原家族(CA125、CA15-3等)等。
然而，在癌症确诊阶段，以上临床应用的常规影像学所检测到的恶性肿瘤，直径一般在1em以上，肿瘤细胞的数目达到109之多，重量超过1g。PET-CT灵敏度高，可以检测的肿瘤也要达到0.5cm以上(H.Li et al.2013；步召德，薛钟麒等，2000)。也就是说，影像学检测到的肿瘤患者，大多数属于中晚期，已经错过了癌症治疗的最佳时期。在癌症早期，即便检测到肿瘤标记物，影像学检测不到肿瘤组织的具体位置，也无法最终确诊肿瘤，而进行及早治疗用药，只能任其发展恶化。
肿瘤的耐药性和治愈后的复发是导致肿瘤患者死亡的最重要原因之一，破解这两方面的难题已经成为当今世界肿瘤疾病的研究热点。癌症治疗过程中，目前的临床应用缺乏一种有效的用药评估手段。以肺癌为例，吉非替尼(易瑞沙)、厄洛替尼(特罗凯)是非小细胞肺癌(NSCLC)的治疗药物，而临床数据表明并不是所有的非小细胞肺癌患者都适合使用此类药物。进一步研究发现带有EGFR(epidermal growth factor receptor)突变的患者接受易瑞沙治疗反应率比无选择性的患者高40％以上(Tony S.Mok，M.D.et al.2009；Hida T，Okamoto I，Kashii T，et al.2008；Kimura H，Kasahara K，Kawaishi M，et al.2006)。吉西他滨是一种破坏细胞复制的二氟核苷类抗代谢物抗癌药，适用于治疗中、晚期非小细胞肺癌。研究证实：RRM1(RRM1基因定位于1号染色体短臂，编码核糖核苷酸还原酶M1亚单位)表达量低的患者接受吉西他滨治疗疾病控制率高30％以上(Lee et al.2010)。欧洲药监局明确规定，非小细胞肺癌患者在使用易瑞沙前必须检测EGFR基因，转移性大肠癌患者在使用靶向药物爱必妥和维克替比治疗前必须检测KRAS基因(来自欧洲药监局官方网站http：//www.ema.europa.eu/ema/)。由此可见，对于恶性肿瘤的治疗，用药指导评估是优化治疗方案，实现有效治疗的重要前提和基础。肿瘤发生、发展的系统生物学研究得出结论，DNA突变或者由遗传获得的缺陷基因是导致耐药性的关 键，因此，从基因水平进行个体化用药指导和评估，大势所趋。
除此之外，临床治疗癌症的预后，也存在检测盲期，例如：很多癌症患者通过手术切除肿瘤组织，以防止其继续扩散而危及生命，术后需定期活检复查进行预后评估。因影像学检测辐射大、对人体造成伤害，癌症患者术后多以年为单位进行复查，这大大影响了预后评估的时效性，同时也反映出癌症诊断领域迫切需要一种更加简便、具无创伤性的检测技术。总而言之，目前临床医学对于早期癌症的筛查、诊断、治疗和预后，是癌症治疗的重大突破口，也是提高癌症五年生存率的关键所在。无论是医学研究，还是推广到临床应用，这一领域都存在重大的技术难题。
1947年，Mandel和Metais在血液、滑膜液和脑脊液等体液中，发现了一种胞外DNA，主要以DNA蛋白质复合物或者游离DNA两种形式存在(Mandel&Metais.1947)。到了80年代，Leon等人研究发现，肿瘤患者外周血清DNA水平大大高于正常人(Leon et al.1977)。且研究发现，在肿瘤患者的血浆和血清中检测到了与原发肿瘤相一致的癌基因突变。这表明，循环DNA作为一种新的肿瘤标志物，将在肿瘤的诊断、治疗和预后检测等领域发挥重要作用。循环肿瘤DNA(circulating tumor DNA，ctDNA)是肿瘤细胞体细胞DNA经脱落或者当细胞凋亡后释放进入循环系统，可以被定性、定量和追踪。对于这类ctDNA的成功捕获，携带信息的准确解读为癌症早期基因信息的获取，癌症的早期诊断、预后检测、耐药评估提供了一条准确的途径。
但是，这类技术目前仍未得到有效解决和广泛应用。原因之一在于：ctDNA在外周血中的含量非常低，尤其是与正常DNA(normal DNA，nDNA)相比，其相对含量极低，目前的检测技术还难以达到直接检测外周血中ctDNA的水平。因此，需要先通过DNA富集的方法将目标ctDNA富集起来，然后在通过对富集的ctDNA进行测序，得到其携带信息，并根据测序得到的携带信息来解读个体的患病情况。
发明内容
本发明提供一种富集循环肿瘤DNA的方法和试剂，该方法能够有效地捕获外周血液血浆中的ctDNA，并且进行单分子高保真超微量平行扩增，提供了足够量ctDNA以进行后续的测序检测使用。
根据本发明的第一方面，本发明提供一种富集循环肿瘤DNA的方法，包括如下步骤：
单分子平行扩增：将水相和油相混合、振荡配成乳液PCR反应体系并进行乳液PCR扩增，其中所述水相中含有作为模板DNA的外周血液血浆DNA、正反向引物、dNTPs、PCR缓冲液和DNA聚合酶，所述外周血液血浆DNA的两端连有接头序列，所述正反向引物分别与两端的接头序列互补配对；
分离水相和油相：所述乳液PCR扩增后，分离水相和油相，得到水相的PCR扩增产物；
捕获循环肿瘤DNA：使用特异性结合循环肿瘤DNA的探针序列捕获所述水相的PCR扩增产物中的循环肿瘤DNA。
作为本发明的优选方案，所述乳液PCR反应体系的水相中还含有dUTP；
优选地，所述dUTP与所述dNTPs的摩尔比为1∶1000～1∶10，更优选为1∶100。
作为本发明的优选方案，所述接头序列为测序设备中使用的测序接头序列；
优选地，所述测序设备为Illumina测序仪，所述测序接头序列对应的正反向引物序列分别为5’-TCCCTACACGACGCTCTTCCGATCT-3’(SEQ ID NO：1)和5’-TGAACCTGAACCGCTCTTCCGATCT-3’(SEQ ID NO：2)；
优选地，所述测序设备为Life Tech测序仪，所述测序接头序列对应的正反向引物序列分别为5’-CCATCTCATCCCTGCGTGTCTCCGA-3’(SEQ ID NO：3)和5’-CCGCTTTCCTCTCTATGGGCAGTCG-3’(SEQ ID NO：4)。
作为本发明的优选方案，所述水相与油相的体积比为1∶10～1∶1，优选为1∶5～1∶2。
作为本发明的优选方案，所述DNA聚合酶为高保真DNA聚合酶，优选为高保真的Klenow Fragment(DNA聚合酶I，DNAPolymerase I)、KAPAHiFi家族高保真DNA聚合酶、Phusion家族高保真DNA聚合酶或Q5家族高保真DNA聚合酶。
作为本发明的优选方案，所述水相中外周血液血浆DNA总量1-100ng、正反向引物终浓度为0.1-1μM、dNTPs终浓度为0.5-2mM、PCR缓冲液终浓度为1倍、DNA聚合酶终浓度为0.1-1U；
优选地，所述水相中外周血液血浆DNA总量1-10ng、正反向引物终浓度为0.5μM、dNTPs终浓度为1mM、PCR缓冲液终浓度为1倍、DNA聚合酶终浓度为0.25U；
优选地，所述乳液PCR扩增的程序为93-95℃1-3min；93-95℃5-20s，61-65℃ 10-20s，71-73℃5-20s，40-60个循环；71-73℃4-10min；
优选地，所述乳液PCR扩增的程序为94℃2min；94℃10s，63℃15s，72℃10s，50个循环；72℃5min。
作为本发明的优选方案，所述捕获循环肿瘤DNA中使用的特异性结合循环肿瘤DNA的探针序列带有生物素修饰；所述探针序列与所述循环肿瘤DNA特异性结合以后，通过链霉亲和素磁珠特异性结合生物素而捕获所述循环肿瘤DNA；需要说明的是：生物素修饰可以在探针序列的任意位置，比如探针序列的5’端、3’端或中间任意碱基位置。
优选地，在使用所述探针序列捕获所述循环肿瘤DNA过程中，使用封闭序列封闭所述接头序列，其中所述封闭序列特异性结合外周血液血浆DNA两端的接头序列。
作为本发明的优选方案，所述捕获循环肿瘤DNA，具体通过使用特异性结合循环肿瘤DNA的探针序列作为引物，进行PCR扩增而实现，其中所述作为引物的探针序列为特异性结合循环肿瘤DNA的序列。
根据本发明的第二方面，本发明提供一种富集循环肿瘤DNA的试剂，所述试剂包括如下组成部分：
乳液PCR扩增组分，包括水相和油相，其中所述水相包含正反向引物、dNTPs、PCR缓冲液和DNA聚合酶，所述正反向引物分别与连接于外周血液血浆DNA两端的接头序列互补配对；
捕获循环肿瘤DNA组分，包括特异性结合循环肿瘤DNA的探针序列，用于捕获所述水相的PCR扩增产物中的循环肿瘤DNA；
优选地，所述乳液PCR扩增组分的水相中还含有dUTP；
优选地，所述dUTP与所述dNTPs的摩尔比为1∶1000～1∶10，更优选为1∶100：
优选地，所述DNA聚合酶为高保真DNA聚合酶，优选为高保真的Klenow Fragment(DNA Polymerase I)、KAPA HiFi家族高保真DNA聚合酶、Phusion家族高保真DNA聚合酶或Q5家族高保真DNA聚合酶。
作为本发明的优选方案，所述探针序列带有生物素修饰，并且所述试剂还包括链霉亲和素磁珠，所述探针序列与所述循环肿瘤DNA特异性结合以后，通过链霉亲和素磁珠特异性结合生物素而捕获所述循环肿瘤DNA；
优选地，所述试剂还包括封闭序列，所述封闭序列特异性结合外周血液血 浆DNA两端的接头序列，用于在使用所述探针序列捕获所述循环肿瘤DNA过程中封闭所述接头序列；
优选地，所述探针序列为特异性结合循环肿瘤DNA的序列，用于进行PCR扩增得到富集的循环肿瘤DNA。
本发明的方法通过乳液PCR与探针捕获技术的结合，实现单分子高保真超微量平行扩增以及有效地捕获外周血液血浆中的ctDNA，提供了足够量ctDNA以进行后续的测序检测使用。与传统的病理学检测方法相比，本发明的方法无需组织细胞活检取样，只要少量外周血液血浆即可大量富集ctDNA，用于后续的测序检测，因此实现了无创检测。不仅节省了检测费用、提高了诊断准确性，而且打破了癌症的检测极限，由传统的中晚期检测，提前到早期诊断，并且本发明的方法操作简便。
需要说明的是：本发明的富集循环肿瘤DNA的方法本身还不能确定患者的患癌情况，因为该方法本身只在于富集外周血液血浆中的循环肿瘤DNA，为后续测序分析携带信息准备足够的样本材料，因此富集循环肿瘤DNA的方法本身并不能作为诊断方法。
附图说明
图1为本发明中的乳液PCR的原理和过程示意图；
图2为本发明中的生物素-寡核苷酸捕获ctDNA的原理和过程示意图；
图3为本发明实施例1捕获得到的ctDNA凝胶电泳检测结果；
图4为本发明中的引物PCR捕获ctDNA的原理和过程示意图；
图5为本发明实施例2捕获得到的ctDNA凝胶电泳检测结果；
图6为本发明实施例3捕获得到的ctDNA凝胶电泳检测结果。
具体实施方式
下面通过具体实施例对本发明作进一步详细说明。除非特别说明，下面实施例中所使用的技术均为本领域内的技术人员已知的常规技术；所使用的仪器设备和试剂等，均为本领域内的技术人员可以通过公共途径如商购等获得的。
本发明主要解决了以下两个问题：
1、ctDNA和nDNA的平行扩增问题：由于ctDNA与nDNA的比例约为1/1000，如何有效地平行扩增是决定评判结果的关键。传统的PCR扩增方法的结果是nDNA随扩增进行所占比例越来越大，而ctDNA随扩增进行所占比例越来越小。本发明的方法，对外周血液血浆中的DNA(包含ctDNA和nDNA)进 行超微量平行扩增，确保扩增后，两者比例仍为1/1000。
2、ctDNA的捕获问题：对于上面平行扩增的DNA中包含ctDNA和nDNA，需要从全基因组中捕获出癌症基因部分，得到ctDNA，本发明的方法通过捕获ctDNA的探针序列成功实现ctDNA捕获富集。
本发明的方法能够有效捕获ctDNA，进行单分子高保真扩增，为后续检测提供足够量的ctDNA。本发明的方法主要包括两部分，分别针对上述两个问题提出了解决方案。
第一部分，是针对第一个问题的解决方案：用乳液PCR(emulsion PCR，ePCR)单分子平行扩增ctDNA和nDNA。
第二部分，是针对第二个问题的解决方案：用生物素标记的寡核苷酸(biotin-oligo)杂交癌症基因，然后结合链霉亲和素磁珠(streptavidin magnetic beads)，捕获分离出癌症基因；或者设计引物，PCR扩增ctDNA，捕获分离出癌症基因。
本发明分两部分列出具体技术方案，如下：
第一部分：乳液PCR
请参考图1，乳液PCR(emulsion PCR，ePCR)是利用油包水乳液体系中的微液滴作为反应器进行PCR扩增。ePCR体系包括油相和水相的反应体系。油相作为载体，本发明中使用的油相可以不作限定，优选可以采用表1所示的油相体系。
表1本发明优选采用的油相配方体系
Figure PCTCN2015070188-appb-000001
水相中反应体系中包括：DNA模板(DNAtemplate)、dNTPs(包括dATP、dTTP、dCTP、dGTP)、PCR缓冲液、DNA聚合酶(DNA polymerase)和双蒸水(ddH2O)。其中，DNA模板选取外周血液血浆中的DNA，其中包含双链的 ctDNA和nDNA。在进行ePCR之前，DNA双链进行三步修饰，第一步，对双链DNA末端进行修复；第二步将“A”加入到DNA片段的3’末端；第三步，在DNA末端加上特定的接头(adapter)，用于后续的测序，接头的选取需结合具体的测序设备(常用的测序仪器如illumina，Life Tech等)。
研究发现，在水相体系中加入少量dUTP，有利于反应进行，其中dUTP与dNTPs的摩尔比为1∶1000～1∶10较佳，更优选为1∶100。
选用illumina系统测序仪时，接头引物部分序列如下：
正向引物(5’-3’)：TCCCTACACGACGCTCTTCCGATCT(SEQ ID NO：1)；反向引物(5’-3’)：TGAACCTGAACCGCTCTTCCGATCT(SEQ ID NO：2)。
选用Life Tech系统测序仪时，接头引物部分序列如下：
Ion_A(Red，5’-3’)：CCATCTCATCCCTGCGTGTCTCCGA(SEQ ID NO：3)；Ion_P1(Blue，5’-3’)：CCGCTTTCCTCTCTATGGGCAGTCG(SEQ ID NO：4)。
除此之外，水相反应体系中的其他组分dNTPs、dUTP、缓冲液(buffer)、DNA聚合酶可以提前配置为Master Mix，见表2，分装后置于-20℃冻存待用。
表2Master Mix配置比例(总体积为200μL)
试剂 量 终浓度(/μL)
10×buffer(含有Mg2+) 20μL 1×
phi29polymerase(10U/μL) 5μL 0.25U
dNTPs(10mM) 20μL 1.0mM
dUTP(0.1mM) 20μL 10μM
ddH2O 135μL -
ePCR中，水相/油相可选取不同的比例，如：75μL/400μL；240μL/960μL；200μL/400μL；300μL/400μL等。以水相/油相比例200μL/400μL为例，油相选取配方2，400μL油相中，加入160μL硅油AR20、120μL环甲基硅氧烷(及)硅酮多元醇共聚物(5225C Formulation Aid)、120μL 749Fluid。水相中ePCR体系及循环参数如表3和表4。
表3PCR体系各组分比例
试剂 量
Master Mix 199μL
DNA模板 1μL(≥1ng)
ddH2O 补足200μL
表4PCR循环参数
Figure PCTCN2015070188-appb-000002
水相和油相依次加入1.5mL不沾壁(non-stick)的反应管中。两相混合后，将装有反应体系的反应管放在组织破碎仪(Qiagen Tissue Lyser II)中震荡90秒，参数设为13Hz。取下反应试管，将油水体系分装至PCR反应管，根据表4的设置参数进行PCR反应。
接下来，PCR结束后，分离PCR反应管中的水相和油相。本发明可以采用三种分离方法，方法一：去掉反应管上部的油相，向反应管中加入400μL石蜡油，涡旋或者用移液管反复吸取管中溶液，直至水相的乳球破裂，水相和油相充分混合。将混合均匀的液体从PCR反应管转移到新的不沾壁反应管中，用400μL硅油(silicon oil)洗脱PCR反应管，洗脱液加入上述水相/油相混合液，充分涡旋，15000rpm离心2min，去掉上层油相。接下来需要对混合液进行洗脱。硅油洗脱三次，加入500μL硅油，充分涡旋，15000rpm离心1min弃上层油相，再重复2次操作。分离的到水相的PCR反应产物。方法二：收集反应管中的水相和油相，9000g离心5min，去掉油相，此时水相仍然呈乳球状沉淀在反应管底部，加入400μL(两倍体积)的饱和乙醚，混合物充分涡旋，15000rpm离心2min，去掉上层的乙醚；乙醚重复洗涤一次，得到水相的PCR反应产物。方法三：收集反应管中的水相和油相，15000rpm离心10min弃上层油相，加入400μL(两倍体积)的用苯酚/三氯甲烷，混合物充分涡旋，15000rpm离心2min，去掉上层的苯酚/三氯甲烷；苯酚/三氯甲烷重复洗涤一次，得到水相的PCR反应产物。
PCR聚合酶，一般选用高保真的Klenow Fragment(DNA Polymerase I)、KAPA HiFi家族、高保真的Phusion家族以及高保真的Q5家族DNA聚合酶等。需要注意的是，得到的DNA产物是两端含有接头的单链DNA，为避免在捕获ctDNA 时，DNA末端的接头发生互补而引入nDNA，得到的ePCR产物在进行后续实验时，加入与接头互补的寡核苷酸序列，即：最后得到的DNA单链，在末端是双链接头形式。且体系中有DNA聚合酶和相应的缓冲液，在进行下一步实验时，考虑是否需要将酶失活(一般65-70℃，10min即可，具体温度根据使用的酶而定)。
第二部分：第一部分得到单链DNA，在此要捕获出ctDNA，有两种方案，分别列出如下。
方案一：请参考图2，主要是通过磁珠特异性的生物素标记的寡核苷酸(biotin-oligo)，捕获出单链ctDNA。此方案过程主要是：结合-洗涤-洗脱。
生物素标记的寡核苷酸，包括两部分：基因互补配对部分和生物素标记部分。生物素可以特异性吸附在链霉亲和素磁珠表面，通过分离磁珠，洗涤去掉其它组分并洗脱，即可分离出ctDNA；基因互补配对部分在捕获ctDNA的过程中与ctDNA进行碱基互补配对结合。
为提高捕获ctDNA效率，基因互补配对部分的长短可根据具体情况调整，一般50bp左右。
在超微量平行扩增的DNA产物中，加入设计好的生物素标记的寡核苷酸，98℃放置5min，60℃震荡2小时，然后冷却至室温。加入混匀的洗净的链霉亲和素磁珠，室温下震荡30min。磁珠必须在液面以下，确保磁珠与含有生物素标记的ctDNA-生物素-寡核苷酸(ctDNA-Biotin-oligo)复合物充分结合。接下来，对磁珠进行洗脱。首先，置于磁力架放置2min，磁珠紧贴在管壁上，去掉液体，再重复两次此操作，直至液体去除干净；加入300μL 1×B&w缓冲液洗涤，快速涡旋，置于磁力架放置2min，磁珠紧贴在管壁上，去掉液体，再重复一次此操作；加入300μL 1×TE缓冲液洗涤，快速涡旋，置于磁力架放置2min，磁珠紧贴在管壁上，去掉液体，再重复一次此操作；最后，加入300μL 0.125M的NaOH溶液，涡旋1sec，在室温中放置10min，然后充分涡旋置于磁力架放置2min，磁珠紧贴在管壁上，取清液，即得到捕获出的ctDNA。
磁珠用1×TE缓冲液洗脱三次后，可重复使用。具体操作如下：第一次洗脱，加入300μL 1×TE缓冲液，涡旋后快速离心，置于磁力架放置30sec，磁珠紧贴在管壁上，去掉上清；第二次洗脱，加入300μL 1×TE缓冲液(含0.01％Trition)涡旋后快速离心，置于磁力架放置30sec，磁珠紧贴在管壁上，去掉上清；第三次洗脱，加入300μL 1×TE缓冲液(含0.01％Trition)，涡旋后快速离心，置于磁 力架放置30sec，磁珠紧贴在管壁上，去掉上清；最后，磁珠用50μL1×TE缓冲液(含0.01％Trition)悬浮，即可。
实施例1：
肺癌患者血样储存在促凝采血管中，取上清1mL，置于1.5mL的EP管中，13000rpm，离心1min，弃沉淀，上清备用。
取上清液的血清400μL，提取DNA。采用过柱回收试剂盒进行(TIANGEN的血清/血浆游离DNA提取试剂盒)，具体操作：1，加入40μL Proteinase K溶液，充分斡旋混匀；2，加入400μL GB缓冲液，2μL carrier RNA(1ng/μL)，混匀，58℃温浴10分钟；3，加入400μL ethyl alcohol，置于室温放置10分钟；4，CR2柱放在2mL的离心管中，将上述溶液转移至CR2柱，12000rpm离心30sec，倒掉管中的废液；5，加入1000μL GD缓冲液，12000rpm离心30sec，弃废液；6，加入1200μL GD缓冲液，12000rpm离心30sec，弃废液；7，重复第6步操作；8，将CR2置于离心机，12000rpm离心2min，室温下放置5min；8，40μL TB缓冲液洗脱CR2，室温下放置5min，然后12000rpm离心2min，为充分洗脱，此步可重复操作一遍。
提取的血清血浆DNA测浓度为6.74ng/μL。
提取的血清血浆DNA进行ePCR，水相/油相比例为200μL/400μL。油相根据表1的配方3进行制备，1mL油相中，700μL乳化剂ABIL WE90、200μL石蜡油，730μL碳酸二乙基己酯。
水相体系中的组分dNTPs、dUTP、缓冲液(buffer)、DNA聚合酶根据表2配置为Master Mix，其中10X缓冲液20μL，phi29DNA聚合酶(10U/μL)5μL，dNTPs(10mM)20μL，dUTP(0.1mM)20μL，灭菌ddH2O 134μL，共199μL。DNA模版加1μL，因此，水相共200μL。
水相200μL和油相400μL，依次加入1.5mL不沾壁的反应管中。两相混合，反应管放在组织破碎仪(Qiagen Tissue Lyser II)中震荡90秒，参数设为13Hz。然后将600μL反应体系分装于6个反应管中，100μL/管，进行ePCR。
PCR参数设置三步操作，第一步，预变性，94℃，2min；第二步循环操作，变性，94℃，10sec，引物退火，63℃，15sec，延伸72℃，10sec，共进行35个循环；第三步，终延伸，72℃，5min。得到的ePCR反应产物置于4℃保存备用即可。
得到ePCR的产物进行后期处理，分离水相和油相。具体操作：1，去掉反 应管上部的油相，向反应管中加入400μL石蜡油，涡旋或者用移液管反复吸取管中溶液，直至水相的乳球破裂，水相和油相充分混合。2，洗脱反应管，将混合均匀的液体转移到新的不沾壁反应管中，用400μL硅油洗脱PCR反应管，洗脱液加入上述水相/油相混合液，充分涡旋，15000rpm离心2min，去掉上层油相。3，洗脱水相/油相混合液。硅油洗脱三次，加入500μL硅油，充分涡旋，15000rpm离心1min弃上层油相，再重复2次操作。用移液枪将水相的液体，轻轻吸出，转移至新的EP管中，得到水相的PCR反应产物200μL。
EPCR扩增的DNA产物中，含有DNA聚合酶，因此，在进行下一步实验之前，要进行DNA聚合酶失活，将反应管于65℃，温浴10min。
从ePCR得到的DNA产物，捕获ctDNA，设计生物素标记的寡核苷酸(Biotin-oligo)。待捕获的EGFR第17个外显子(exon 17th)序列如下(5’-3’)：
GCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTT GCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGCGAAGGCGCCACATCGTTCGGAAGCGCACGCTGCGGAGGCTGCTGCAGGAGAGGGAG(SEQ ID NO：5)。
设计基因互补配对部分(与下划线部分配对结合)：
5’-/Bio/GAGGCCGATCCCCAGGGCCACCACCAGCAGCAAGAGGAG
GGCCCCCACCA-3’(SEQ ID NO：6)，其中5’-端部分带有生物素标记。
在ePCR的DNA产物中，加入5’-端部分带有生物素标记的寡核苷酸，捕获ctDNA。具体操作：1，200μL水相的PCR反应产物，加入生物素标记的寡核苷酸20μL(寡核苷酸浓度10μM，终浓度1μM)，98℃放置5min，60℃震荡2小时，然后冷却至室温。2，加入50μL混匀的洗净的链霉亲和素磁珠，室温下震荡30min。磁珠必须在液面以下，确保磁珠与含有生物素标记的ctDNA-生物素-寡核苷酸(ctDNA-Biotin-oligo)复合物充分结合。3，吸附磁珠。将反应管置于磁力架放置2min，磁珠紧贴在管壁上，去掉液体，再重复两次此操作，直至液体去除干净。4，洗脱磁珠。加入300μL 1×B&w缓冲液洗涤，快速涡旋，置于磁力架放置2min，磁珠紧贴在管壁上，去掉液体，再重复一次此操作；加入300μL1×TE缓冲液洗涤，快速涡旋，置于磁力架放置2min，磁珠紧贴在管壁上，去掉液体，再重复一次此操作；4，加入300μL 0.125M的NaOH溶液，涡旋1sec，在室温中放置10min，然后充分涡旋置于磁力架放置2min，磁珠紧贴在管壁上，取清液，即得到捕获出的ctDNA。
捕获的DNA产物，大小为142bp，取5μL捕获的DNA溶液，加入1μL6X上样缓冲液(loading buffer)，充分混匀，点样进行电泳检测，DNA marker选用100bp plus(GENSTAR)，上样1μL。琼脂糖凝胶配置的浓度为2％，取琼脂糖2g，加入100mL 1X TAE缓冲液中，微波加热2min，至琼脂糖完全溶解，待溶液冷却至50-60℃，加入5μL核酸染料(Gold View)，倒胶。
电泳检测结果如图3所示。其中，捕获的DNA溶液上样至第11和第12泳道，根据电泳图分析，目的条带大小符合，即该方法成功捕获得到ctDNA，可用于后续其他检测。
方案二：请参考图4，主要是通过引物PCR扩增获得ctDNA。上述超微量平行扩增的DNA产物中，含有DNA聚合酶，因此，在进行方案二时要进行DNA聚合酶失活，65℃，10min处理，即可。
设计PCR引物，包括两部分：基因互补配对部分和接头(adapter)部分。所述PCR引物无需末端修饰，基因互补配对部分位于3’端，PCR扩增时与单链ctDNA发生碱基配对；接头部分位于5’端，在后续的基因测序中发挥作用。
为提高捕获ctDNA效率，基因互补配对部分的长短可根据具体情况调整，一般25bp左右，不低于20bp；在接头部分设计时，结合具体的测序设备选择序列，长度一般不低于20bp。
PCR的合成酶，一般选用高保真的Klenow Fragment(DNA Polymerase I)、KAPAHiFi家族、高保真的Phusion家族以及高保真的Q5家族DNA聚合酶等。因此，在设计引物时，引物退火温度应当符合所选合成酶的活性温度。
实施例2：
实施例1中，ePCR扩增的DNA产物中，含有DNA聚合酶，因此，在进行下一步实验之前，要进行DNA聚合酶失活，将反应管于65℃，温浴10min。
EPCR扩增得到200μL DNA产物，采用引物PCR捕获ctDNA。设计PCR引物，待捕获的EGFR第17个外显子(exon 17th)序列如下(5’-3’)：
GCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGCGAAGGCGCCACATCGTTCGGAAGCGCACGCTGCGGAGGCTGCTGCAGGAGAGGGAG(SEQ ID NO：5)。
基因互补配对部分(下划线部分)：
正向引物：(5’-3’)GCCTAAGATCCCGTCCATCGCCA(SEQ ID NO：7)；
反向引物：(5’-3’)CTCCCTCTCCTGCAGCAGCCTC(SEQ ID NO：8)。
接头部分(选用illumina系统测序仪)：
正向引物：(5’-3’)TCCCTACACGACGCTCTTCCGATCT(SEQ ID NO：1)；
反向引物：(5’-3’)TGAACCTGAACCGCTCTTCCGATCT(SEQ ID NO：2)。
因此，设计的引物如下，Tm表示退火温度(盐浓度为50mM)：
正向引物：
TCCCTACACGACGCTCTTCCGATCTGCCTAAGATCCCGTCCATCGCCA(SEQ ID NO：9，Tm：69.7℃)；
反向引物：
TGAACCTGAACCGCTCTTCCGATCTCTCCCTCTCCTGCAGCAGCCTC(SEQ ID NO：10，Tm：68.5℃)。
ePCR得到的DNA产物200μL，测浓度为50ng/μL，取1μL稀释10倍，得到浓度为5ng/μL的DNA溶液。以此浓度的DNA溶液为模板进行PCR扩增。
PCR扩增体系和循环参数如下表4和表5所示。合成酶为KAPA2G Robust HotStart。
表4PCR体系各组分比例
Figure PCTCN2015070188-appb-000003
表5PCR循环参数
Figure PCTCN2015070188-appb-000004
PCR产物，大小为192bp(含有引物两端的adapter)，取5μLPCR反应产物，加上1μL 6X上样缓冲液(loading buffer)，充分混匀，点样进行电泳检测，DNA marker选用100bp plus(GENSTAR)，上样1μL。琼脂糖凝胶的浓度为2％，配置同于实施例1。
电泳检测结果如图5所示。其中，捕获的DNA溶液上样至第9泳道，根据电泳图分析，目的条带大小符合，即该方法成功捕获得到ctDNA。为进一步检测确定，该PCR获得的DNA是EGFR第17个外显子，对PCR产物进行一代测序。
首先，扩大体系进行核酸胶电泳，琼脂糖凝胶配置的浓度为2％，进行胶回收。剩余的95μL PCR反应产物，加上19μL6X上样缓冲液(loading buffer)，充分混匀，点样进行电泳检测，DNA marker选用100bp plus(GENSTAR)，上样6μL。琼脂糖凝胶的制备方法同上。
然后，对上述PCR产物进行胶回收(OMEGA胶回收试剂盒)，具体操作：1，紫外下切下含有目的条带EGFR基因exon17的核酸胶，称重0.50000g(空白EP管重1.04100g，加入切割的胶体重1.54100g)；2，加入binding buffer XP2500μL，55-65℃温浴7min，直至核酸胶完全溶解，混匀；3，将上述溶液转移至干净的离心柱中，离心柱置于新的2mL离心管中，10000g离心1min，弃废液；4，离心柱中加入300μL的binding buffer XP2，10000g离心1min，弃废液；5，加入700μL的wash buffer SPW，13000g离心2min，弃废液；6，重复步骤5；7，离心柱放回离心管中，13000g离心2min，弃废液；8，离心柱在室温下放置约5min，直至干燥；9，离心柱放入干净的EP管中，30μL灭菌的ddH2O，室温下放置1min，13000g离心1min，为充分洗脱，此步可重复操作一遍。
最后，将回收产物(30ng/μL，≥10μL)送至生工进行一代测序，正向引物测序。
从序列比对的结果分析，PCR产物序列与EGFR基因的exton17序列比对正确。但是，从测序结果的碱基峰图看出，1-50bp DNA碱基的峰值出现套封。血清提取的DNA中包含nDNA和ctDNA，二者比例约为1000∶1，因此出现套峰，表示该部分DNA含有nDNA和ctDNA，ctDNA序列可能存在突变现象。
实施例3：(选用Life Tech系统测序仪)：
实施例1中，ePCR扩增的DNA产物中，含有DNA聚合酶，因此，在进行下一步实验之前，要进行DNA聚合酶失活，将反应管于65℃，温浴10min。
EPCR扩增得到的DNA产物，采用引物PCR捕获ctDNA。设计PCR引物，待捕获的EGFR第17个外显子(exon 17th)序列如下(5’-3’)：
GCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGCGAAGGCGCCACATCGTTCGGAAGCGCACGCTGCGGAGGCTGCTGCAGGAGAGGGAG(SEQ ID NO：5)。
基因互补配对部分(下划线部分)：
正向引物：(5’-3’)GCCTAAGATCCCGTCCATCGCCA(SEQ ID NO：7)；
反向引物：(5’-3’)CTCCCTCTCCTGCAGCAGCCTC(SEQ ID NO：8)。
接头引物部分(选用Life Tech系统测序仪)：
Ion_A(Red，5’-3’)：CCATCTCATCCCTGCGTGTCTCCGA(SEQ ID NO：3)；
Ion_P1(Blue，5’-3’)：CCGCTTTCCTCTCTATGGGCAGTCG(SEQ ID NO：4)。
因此，设计的引物如下：
正向引物(5’-3’)：
CCATCTCATCCCTGCGTGTCTCCGACTCAGGCCTAAGATCCCGTCCATCGCCA(SEQ ID NO：11)；
反向引物(5’-3’)：
CCGCTTTCCTCTCTATGGGCAGTCGGTGATCTCCCTCTCCTGCAGCAGCCTC(SEQ ID NO：12)。
EPCR得到的DNA产物200μL，测浓度为50ng/μL，取1μL稀释10倍，得到浓度为5ng/μL的DNA溶液。以此浓度的DNA溶液为模板进行PCR扩增。
PCR扩增体系和循环参数如下表6和表7所示。合成酶为Phusion DNA聚合酶(Phusion DNA Polvmerase)。
表6PCR体系各组分比例
Figure PCTCN2015070188-appb-000005
Figure PCTCN2015070188-appb-000006
表7PCR循环参数
Figure PCTCN2015070188-appb-000007
PCR产物，大小为192bp(含有引物两端的adapter)，取5μL PCR反应产物，加上1μL 6X上样缓冲液(loading buffer)，充分混匀，点样进行电泳检测，DNA marker选用100bp plus(GENSTAR)，上样1μL。琼脂糖凝胶的浓度为2％，制备方法同于实施例1。
电泳检测结果如下图6所示。其中，捕获的DNA溶液上样至第10泳道，根据电泳图分析，目的条带大小符合，即，该方法成功捕获得到ctDNA。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换。

Claims (10)

  1. 一种富集循环肿瘤DNA的方法，其特征在于，所述方法包括如下步骤：
    单分子平行扩增：将水相和油相混合、振荡配成乳液PCR反应体系并进行乳液PCR扩增，其中所述水相中含有作为模板DNA的外周血液血浆DNA、正反向引物、dNTPs、PCR缓冲液和DNA聚合酶，所述外周血液血浆DNA的两端连有接头序列，所述正反向引物分别与两端的接头序列互补配对；
    分离水相和油相：所述乳液PCR扩增后，分离水相和油相，得到水相的PCR扩增产物；
    捕获循环肿瘤DNA：使用特异性结合循环肿瘤DNA的探针序列捕获所述水相的PCR扩增产物中的循环肿瘤DNA。
  2. 根据权利要求1所述的富集循环肿瘤DNA的方法，其特征在于，所述乳液PCR反应体系的水相中还含有dUTP；
    优选地，所述dUTP与所述dNTPs的摩尔比为1∶1000～1∶10，更优选为1∶100。
  3. 根据权利要求1所述的富集循环肿瘤DNA的方法，其特征在于，所述接头序列为测序设备中使用的测序接头序列；
    优选地，所述测序设备为Illumina测序仪，所述测序接头序列对应的正反向引物序列分别为5’-TCCCTACACGACGCTCTTCCGATCT-3’(SEQ ID NO：1)和5’-TGAACCTGAACCGCTCTTCCGATCT-3’(SEQ ID NO：2)；
    优选地，所述测序设备为Life Tech测序仪，所述测序接头序列对应的正反向引物序列分别为5’-CCATCTCATCCCTGCGTGTCTCCGA-3’(SEQ ID NO：3)和5’-CCGCTTTCCTCTCTATGGGCAGTCG-3’(SEQ ID NO：4)。
  4. 根据权利要求1所述的富集循环肿瘤DNA的方法，其特征在于，所述水相与油相的体积比为1∶10～1∶1，优选为1∶5～1∶2。
  5. 根据权利要求1所述的富集循环肿瘤DNA的方法，其特征在于，所述DNA聚合酶为高保真DNA聚合酶，优选为高保真的Klenow Fragment、KAPA HiFi家族高保真DNA聚合酶、Phusion家族高保真DNA聚合酶或Q5家族高保真DNA聚合酶。
  6. 根据权利要求1所述的富集循环肿瘤DNA的方法，其特征在于，所述水相中外周血液血浆DNA总量1-100ng、正反向引物终浓度为0.1-1μM、dNTPs终浓度为0.5-2mM、PCR缓冲液终浓度为1倍、DNA聚合酶终浓度为0.1-1U；
    优选地，所述水相中外周血液血浆DNA总量1-10ng、正反向引物终浓度为 0.5μM、dNTPs终浓度为1mM、PCR缓冲液终浓度为1倍、DNA聚合酶终浓度为0.25U；
    优选地，所述乳液PCR扩增的程序为93-95℃1-3min；93-95℃5-20s，61-65℃10-20s，71-73℃5-20s，40-60个循环；71-73℃4-10min；
    优选地，所述乳液PCR扩增的程序为94℃2min；94℃10s，63℃15s，72℃10s，50个循环；72℃5min。
  7. 根据权利要求1所述的富集循环肿瘤DNA的方法，其特征在于，所述捕获循环肿瘤DNA中使用的特异性结合循环肿瘤DNA的探针序列带有生物素修饰；所述探针序列与所述循环肿瘤DNA特异性结合以后，通过链霉亲和素磁珠特异性结合生物素而捕获所述循环肿瘤DNA；
    优选地，在使用所述探针序列捕获所述循环肿瘤DNA过程中，使用封闭序列封闭所述接头序列，其中所述封闭序列特异性结合外周血液血浆DNA两端的接头序列。
  8. 根据权利要求1所述的富集循环肿瘤DNA的方法，其特征在于，所述捕获循环肿瘤DNA，具体通过使用特异性结合循环肿瘤DNA的探针序列作为引物，进行PCR扩增而实现，其中所述作为引物的探针序列为特异性结合循环肿瘤DNA的序列。
  9. 一种富集循环肿瘤DNA的试剂，其特征在于，所述试剂包括如下组成部分：
    乳液PCR扩增组分，包括水相和油相，其中所述水相包含正反向引物、dNTPs、PCR缓冲液和DNA聚合酶，所述正反向引物分别与连接于外周血液血浆DNA两端的接头序列互补配对；
    捕获循环肿瘤DNA组分，包括特异性结合循环肿瘤DNA的探针序列，用于捕获所述水相的PCR扩增产物中的循环肿瘤DNA；
    优选地，所述乳液PCR扩增组分的水相中还含有dUTP；
    优选地，所述dUTP与所述dNTPs的摩尔比为1∶1000～1∶10，更优选为1∶100；
    优选地，所述DNA聚合酶为高保真DNA聚合酶，优选为高保真的Klenow Fragment、KAPA HiFi家族高保真DNA聚合酶、Phusion家族高保真DNA聚合酶或Q5家族高保真DNA聚合酶。
  10. 根据权利要求9所述的富集循环肿瘤DNA的试剂，其特征在于，所述 探针序列带有生物素修饰，并且所述试剂还包括链霉亲和素磁珠，所述探针序列与所述循环肿瘤DNA特异性结合以后，通过链霉亲和素磁珠特异性结合生物素而捕获所述循环肿瘤DNA；
    优选地，所述试剂还包括封闭序列，所述封闭序列特异性结合外周血液血浆DNA两端的接头序列，用于在使用所述探针序列捕获所述循环肿瘤DNA过程中封闭所述接头序列；
    优选地，所述探针序列为特异性结合循环肿瘤DNA的序列，用于进行PCR扩增得到富集的循环肿瘤DNA。
PCT/CN2015/070188 2015-01-06 2015-01-06 一种富集循环肿瘤dna的方法和试剂 WO2016109939A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN201580006707.6A CN106103742B (zh) 2015-01-06 2015-01-06 一种富集循环肿瘤dna的方法和试剂
PCT/CN2015/070188 WO2016109939A1 (zh) 2015-01-06 2015-01-06 一种富集循环肿瘤dna的方法和试剂
US15/127,736 US9944993B2 (en) 2015-01-06 2015-01-06 Method for enrichment of circulating tumor DNA and reagent for enrichment of circulating tumor DNA

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/070188 WO2016109939A1 (zh) 2015-01-06 2015-01-06 一种富集循环肿瘤dna的方法和试剂

Publications (1)

Publication Number Publication Date
WO2016109939A1 true WO2016109939A1 (zh) 2016-07-14

Family

ID=56355402

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/070188 WO2016109939A1 (zh) 2015-01-06 2015-01-06 一种富集循环肿瘤dna的方法和试剂

Country Status (3)

Country Link
US (1) US9944993B2 (zh)
CN (1) CN106103742B (zh)
WO (1) WO2016109939A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107034301A (zh) * 2017-06-07 2017-08-11 深圳市海普洛斯生物科技有限公司 一种检测肺结节为良性或恶性的试剂盒及其应用

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107058528A (zh) * 2017-03-22 2017-08-18 深圳海普洛斯医学检验所有限公司 一种肺癌EGFR T790M位点的ddPCR检测优化方法及应用
WO2018209092A1 (en) * 2017-05-10 2018-11-15 Board Of Regents, The University Of Texas System Methods and devices related to amplifying nucleic acid at a variety of temperatures
CN107119145A (zh) * 2017-07-13 2017-09-01 深圳瑞科生物科技有限公司 一种基于ddPCR定量检测ctDNA的方法
CN108070910A (zh) * 2017-12-11 2018-05-25 上海赛安生物医药科技股份有限公司 cfDNA捕获建库方法
CN110240999B (zh) * 2018-03-09 2022-09-06 浙江品级基因科技有限公司 一种提高循环肿瘤dna检出率的检测装置及方法
CN110456034B (zh) * 2018-05-07 2022-07-22 上海市第十人民医院 一种循环肿瘤细胞的检测方法
CN114901818A (zh) * 2018-11-21 2022-08-12 艾维达生物医学公司 靶向核酸文库形成的方法
CN114292904B (zh) * 2021-12-25 2022-11-01 武汉承启医学检验实验室有限公司 一种优化ctDNA检测准确率的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014028378A2 (en) * 2012-08-13 2014-02-20 The Regents Of The University Of California Methods and systems for detecting biological components
CN104212879A (zh) * 2013-06-05 2014-12-17 上海派森诺生物科技有限公司 一种基于磁珠富集法高通量开发基因组ssr标记的方法

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9399797B2 (en) * 2010-02-12 2016-07-26 Raindance Technologies, Inc. Digital analyte analysis
CN103998625B (zh) * 2011-11-18 2017-06-06 华大生物科技（武汉）有限公司 用于病毒检测的方法和系统
US20150284783A1 (en) * 2012-05-21 2015-10-08 Agena Bioscience, Inc. Methods and compositions for analyzing nucleic acid
WO2014088979A1 (en) * 2012-12-03 2014-06-12 Yilin Zhang Compositions and methods of nucleic acid preparation and analyses
US9982255B2 (en) * 2013-03-11 2018-05-29 Kailos Genetics, Inc. Capture methodologies for circulating cell free DNA
US20130189684A1 (en) * 2013-03-12 2013-07-25 Sequenom, Inc. Quantification of cell-specific nucleic acid markers
EP2971097B1 (en) * 2013-03-15 2018-08-01 Verinata Health, Inc Generating cell-free dna libraries directly from blood
KR102649364B1 (ko) * 2013-11-07 2024-03-20 더 보드 어브 트러스티스 어브 더 리랜드 스탠포드 주니어 유니버시티 인간 마이크로바이옴 및 그의 성분의 분석을 위한 무세포 핵산
CN104263726A (zh) * 2014-09-25 2015-01-07 天津诺禾致源生物信息科技有限公司 适用于扩增子测序文库构建的引物及扩增子测序文库的构建方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014028378A2 (en) * 2012-08-13 2014-02-20 The Regents Of The University Of California Methods and systems for detecting biological components
CN104212879A (zh) * 2013-06-05 2014-12-17 上海派森诺生物科技有限公司 一种基于磁珠富集法高通量开发基因组ssr标记的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107034301A (zh) * 2017-06-07 2017-08-11 深圳市海普洛斯生物科技有限公司 一种检测肺结节为良性或恶性的试剂盒及其应用

Also Published As

Publication number Publication date
US9944993B2 (en) 2018-04-17
US20170067117A1 (en) 2017-03-09
CN106103742A (zh) 2016-11-09
CN106103742B (zh) 2019-12-10

Similar Documents

Publication Publication Date Title
WO2016109939A1 (zh) 一种富集循环肿瘤dna的方法和试剂
CN106047998B (zh) 一种肺癌基因的检测方法及应用
CN108473975A (zh) 检测肿瘤发展的系统和方法
CN107405540A (zh) 小核酸的富集
CN106148498B (zh) Kras基因突变检测试剂盒及其应用
CN107849606A (zh) 提高下一代测序灵敏度的方法
CN109112216A (zh) 三重qPCR检测DNA甲基化的试剂盒和方法
KR101320633B1 (ko) 실시간 중합반응을 이용한 암의 진단을 위한 정보제공방법 및 이를 위한 암 진단용 키트
CN110117652A (zh) 肝癌早期诊断方法
CN105683396B (zh) 用于预测大肠癌发病危险度的基因标志物及其应用
Stelcer et al. Liquid Biopsy in Oligometastatic Prostate Cancer—A Biologist's Point of View
CN111187810A (zh) 非诊断治疗目的的多个肿瘤相关基因的检测方法
CN107312770A (zh) 一种用于高通量测序检测的肿瘤brca1/2基因变异文库的构建方法及其应用
CN106480017A (zh) 同时提取循环肿瘤细胞dna和肿瘤游离dna的试剂盒
KR101955080B1 (ko) 순환 암 바이오마커 및 그의 응용
Chang et al. Liquid biopsy for early diagnosis of non-small cell lung carcinoma: recent research and detection technologies
CN112094916A (zh) 一种血浆游离dna肺癌基因联合检测试剂盒
CN108531586A (zh) 一种与乳腺癌辅助诊断相关的位于X染色体上的循环miRNA标志物及其应用
CN108018353A (zh) 一种基于4个基因的结直肠癌早期筛查引物组及试剂盒
CN106399514A (zh) Nras基因突变检测试剂盒
Shuai et al. Liquid-based biomarkers in breast cancer: looking beyond the blood
CN106967810B (zh) 一种检测fgfr3基因突变以诊断膀胱癌的方法及试剂盒
CN105779433A (zh) 试剂盒及其用途
CN105219841B (zh) 一种肺癌差异性表达microRNA的检测试剂盒及其应用
CN106636442A (zh) 一种人类肿瘤基因变异联合检测试剂盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15876449

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 15127736

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205N DATED 11/12/2017)

122 Ep: pct application non-entry in european phase

Ref document number: 15876449

Country of ref document: EP

Kind code of ref document: A1