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 PDFInfo
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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.
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Abstract
Description
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 | - |
试剂 | 量 |
Master Mix | 199μL |
DNA模板 | 1μL(≥1ng) |
ddH2O | 补足200μL |
Claims (10)
- 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。
- 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。
- 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)。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
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