WO2020151283A1 - 一种基于选择性消除野生链背景干扰的基因突变检测方法 - Google Patents

一种基于选择性消除野生链背景干扰的基因突变检测方法 Download PDF

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WO2020151283A1
WO2020151283A1 PCT/CN2019/113859 CN2019113859W WO2020151283A1 WO 2020151283 A1 WO2020151283 A1 WO 2020151283A1 CN 2019113859 W CN2019113859 W CN 2019113859W WO 2020151283 A1 WO2020151283 A1 WO 2020151283A1
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dna
strand
dnase
target region
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赵美萍
陈维
阳彝栋
肖先金
李梦圆
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北京大学
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  • the present invention relates to the field of genome sample processing and gene mutation detection, in particular to a method for pretreatment of genomic samples. After wild-type DNA is eliminated by this method, it is combined with conventional sequencing methods to realize rapid ultra-low abundance gene mutation , Low-cost sequencing analysis.
  • Cancer is the number one killer threatening human health today. Early diagnosis and postoperative recurrence monitoring related to cancer have important biological and medical significance. Gene mutation refers to the heritable mutation of genomic DNA molecules, which is one of the important reasons for the formation of malignant tumors.
  • the traditional tumor gene detection method is mainly tissue biopsy, that is, tumor tissue samples are obtained through surgical puncture and genetic testing is performed on them. This method is mainly for detecting cancerous tissues. It cannot be effective for early tumor cells that have not yet formed a lesion. For detection, for tumor tissues that have formed lesions, false negative results may also be obtained because of tumor heterogeneity. In addition, this invasive detection method brings greater pain to the patient, and may further stimulate the cancer tissue to cause deterioration during surgical puncture. Tissue biopsy is not suitable for continuous sampling and disease tracking.
  • ctDNA circulating tumor DNA
  • ctDNA 1 circulating tumor DNA
  • circulating tumor cells and exosomes liquid biopsy has gradually become a new hope for early cancer diagnosis.
  • ctDNA is the DNA released into the circulatory system after early tumor cell shedding or apoptosis. This DNA is highly fragmented but carries all the gene mutation information of tumor cells.
  • the detection of ctDNA in the circulating system can provide important information for early tumor screening and recurrence monitoring. Since normal cells also release a large amount of fragmented DNA into the circulatory system during the process of programmed apoptosis, it brings a large degree of background interference to the detection of ctDNA in plasma free DNA (Cell free DNA, cfDNA).
  • the abundance of ctDNA with gene mutations in plasma is generally between 0.001% and 10%, which places high requirements on the sensitivity and selectivity of mutation detection methods.
  • the current detection methods for gene mutations mainly include sequencing, digital droplet PCR, and DNA probe hybridization.
  • the direct sequencing method is the most classic and widely applicable gene mutation detection method.
  • the sequencing method can directly give the nucleotide sequence of the target DNA molecule, so it is regarded as the gold standard for gene mutation detection.
  • the Sanger sequencing method (dideoxy chain termination method) has achieved low-cost and rapid detection of a single sample after decades of development and technical optimization, but its sensitivity is limited, and the minimum detection limit for the abundance of mutant genes is 5%-10% In between, it cannot be used directly for ctDNA detection.
  • Next-generation sequencing (NGS) 2 uses a completely different principle from the classic Sanger chain termination method. It implements Sequencing by synthesis (SBS) in a massively parallel manner.
  • CAPP-NGS Cancer personalized profiling by deep sequencing
  • Nanopore sequencing uses ⁇ -hemolysin to construct biological nanopores, and the middle of the pores just allows single-stranded DNA to pass through. When the DNA strand passes through the nanopore, it will prevent other ions from freely entering and exiting the nanopore, which will cause the current near the nanopore to change.
  • Digital droplet PCR (digital droplet PCR, ddPCR) 4
  • the chip or droplet is used to realize the independent distribution of samples, and PCR is performed on the independent droplets.
  • the limit of detection can reach 0.005-0.05%.
  • the most sensitive method for detecting ctDNA but reaching this limit detection limit requires extremely high requirements for the original PCR system to be ultra-dispersed to droplets.
  • the stable detection limit of this method is artificially controlled between 0.01% and 0.1%. In actual clinical use, the detection limit is controlled between 0.05% and 0.1%. The lower the detection limit, the larger the sample volume required.
  • the key technical core of ddPCR is to realize the single-molecule level dispersion of the DNA to be tested.
  • the actual operation is cumbersome, the instrument is expensive, the operating cost is high, the detection cycle is 2-4 days, and it is not easy to realize the rapid analysis of ctDNA.
  • the fluorescent probe method uses artificially designed and synthesized single-stranded DNA probes labeled with a fluorescent group and a quenching group to specifically recognize and bind to the mutant target chain to be tested in the system and give a fluorescent signal to achieve Detection of the abundance of mutant genes in test samples.
  • the difference between the wild strand and the mutant strand in the sample to be tested is only one base, and the DNA probe has limited distinguishing ability for this base, and the detection limit in the actual application process is about 10%.
  • people have developed a variety of new probes based on simple probes, such as molecular beacons, binary probes, three-stem probes, etc., to reduce the detection limit to 3%.
  • Zhang 5 et al. further introduced a large number of thermodynamic and kinetic calculations on the basis of simple DNA hybridization probes, and used the identification of strand competition, strand displacement, and strand migration to further reduce the detection limit to 0.01% to 1%, but this method detects It takes a long time and requires accurate calculations for probe sequences and competing sequences.
  • Das 6 et al. developed an electrochemical detection method for gene mutations based on probe hybridization without PCR by combining DNA probes and high-sensitivity electrochemical detection methods. This method uses the high sensitivity of electrochemical detection to abandon traditional PCR and can Direct determination of 0.01% of the mutation chain has reached the level of ultra-low abundance gene mutation detection. However, the general applicability of this method is poor.
  • the structure of nanoelectrodes and the synthesis of nanocarriers on the electrode surface are more difficult.
  • the electrochemical detection system needs to be redesigned for different detection systems.
  • the PNA used in the system is expensive, which limits Further promotion of this method.
  • Xiao 7 et al. introduced nuclease-assisted fluorescence signal amplification on the basis of the original fluorescent probe method, which significantly improved the sensitivity of the fluorescent probe method.
  • the fluorescence analysis method lacks direct information of base changes.
  • the existing selective PCR methods mainly include mutation block amplification PCR (Amplification refractory mutation system PCR, ARMS PCR) 8 , wild-type blocking PCR (Wild-type blocking PCR) 9 , low temperature PCR (Co-amplification at lower temperature PCR) , COLD-PCR) 10 , Locked nucleic acids/peptide nucleic acids-mediated PCR (LNA/PNA-mediated PCR) 11, and ARMS-qPCR, ice-COLD PCR, etc. that combine the above methods method.
  • the PCR system itself is a complex biochemical reaction involving multiple temperature combinations and multiple components. The primer design, temperature optimization and time control are all complicated, and the product composition is also very complicated.
  • wild-type DNA does not have mutations, does not carry information about key pathogenic genes, and cannot provide critical information for early tumor screening or postoperative recurrence monitoring, it occupies the vast majority of the detection system, giving the samples of mutant DNA Detection brings huge background interference.
  • a simple and efficient method for selectively removing wild-type DNA in the system can be constructed, the abundance of mutant DNA can be significantly increased, which can then be analyzed by conventional sequencing methods.
  • two research groups have carried out some exploratory studies. They are using double-strand specific nuclease (DSN enzyme) 12 and magnetic bead capture (DISSECT) 13 to selectively remove a large amount of wild Type DNA.
  • DSN enzyme double-strand specific nuclease
  • DISSECT magnetic bead capture
  • the DSN enzyme itself does not have sequence selectivity for wild-type DNA and mutant DNA. Its distinguishing ability comes only from whether the substrate strand is single-stranded or double-stranded, supplemented by precise temperature control. This principle causes the long-stranded DNA of the genome to be non-specifically hydrolyzed, leading to failure of enrichment.
  • Another DISSECT technology uses complementary sequence modified magnetic beads to selectively hybridize to remove wild-type DNA in the system. Because the removal process is linear, the single removal efficiency is low, and the non-specific adsorption on the surface of the magnetic beads can easily lead to the loss of mutant DNA strands.
  • the existing methods still have a series of problems in the early tumor detection for ctDNA, such as insufficient sensitivity, long detection cycle, unstable method, harsh control requirements of experimental conditions, and high price of instrument reagents.
  • the present invention intends to develop a gene mutation detection method with high sensitivity, relatively simple operation, good result stability, low cost, easy to popularize and use and serve for clinical testing.
  • the purpose of the present invention is to provide a gene mutation detection method, by constructing a simple and efficient method to remove wild-type DNA in the system to be tested, so that the abundance of mutant strand DNA can be directly detected by Sanger sequencing method .
  • the sequence of the thioDNA strand to be completely complementary to the wild-type DNA, so that it is rapidly hydrolyzed; while the mutant strand has a single base mismatch with the thioDNA strand, which is basically not Be cut.
  • Genomic samples processed by this system have a significant increase in the proportion of mutant chains.
  • the simplest Sanger sequencing method is used to directly detect whether there are mutations and the types of mutant bases in the genome samples. Further through the quantitative relationship between the processing time and the mutation enrichment factor, the mutation abundance value in the original genome sample can be calculated.
  • the principle of the method of the present invention is shown in Figure 1.
  • the region (10-12 base range) in the mutant DNA strand and the wild-type DNA strand sequence where the mutation site may exist is recorded as the target region, and the thio DNA strand is artificially designed
  • the sequence is completely complementary to the sequence of the target region of the wild-type DNA chain, while the target region of the mutant DNA chain cannot be completely complementary to the sequence of the thio DNA chain due to the existence of the mutation site.
  • the sequence outside the target region of the mutant DNA strand and the wild-type DNA strand is recorded as the non-target region, and the sequences in the non-target region are the same.
  • a single strand of RNA that is completely complementary to its sequence is added to the solution to block it.
  • the above method When the above method is applied to the analysis of genomic samples (such as plasma samples or tissue samples), first use the kit to extract and purify the DNA, and then expand the target sequence (wild type or mutant type) in the extracted DNA by PCR. increase. After the amplified product is processed into single-stranded DNA by Lambda exonuclease, it contains a large amount of wild-type single-stranded DNA and a small amount of mutant-type single-stranded DNA. Add RNA closed strand and thio-DNA strand to the system, add DNase I for cutting after heating and annealing.
  • genomic samples such as plasma samples or tissue samples
  • DNase I selectively excises the target region sequence of the wild-type DNA strand under the guidance of thioDNA, while the mutant DNA strand is retained in the solution system due to mismatches in the target region that cannot be excised by DNase I. As the enzyme digestion reaction proceeds, the abundance of mutant DNA strands increases significantly until the range can be accurately detected by conventional mutation detection methods, which can be directly detected by subsequent PCR amplification and Sanger sequencing.
  • the target sequence includes the target region and the non-target region, design and synthesize the thioDNA strand complementary to the target region of the wild-type DNA sequence and the closed RNA strand complementary to the non-target region;
  • step 3 Mix the single-stranded DNA obtained in step 2) with the thio-DNA strand and RNA closed strand synthesized in step 1) in a DNase I buffer solution. After heating and annealing, add DNase I and react for a period of time to thermally inactivate DNase I;
  • the length of the target sequence to be tested is preferably 115-130 nt, and the target region where a mutation site may be present is preferably located in the middle of the target sequence to be tested. There may be one or more mutation sites in the target region.
  • the length of the target region is controlled at its melting temperature Tm between 42-46°C. Generally, the length of the target region is preferably 11-13 nt. .
  • the non-target region is located at both ends of the target region, and the length of the closed RNA strand complementary to the non-target region is usually 25-90 nt. If the non-target region is relatively long, multiple closed RNA strands can be designed and synthesized.
  • the length of the thio DNA chain is preferably 16-59 nt, including fragments complementary to the wild-type DNA target region and non-complementary fragments at both ends.
  • the thio DNA chain is a full thio DNA chain.
  • the forward primer of the PCR amplification system is the 5'-OH end
  • the 5'end of the reverse primer is phosphorylated
  • the Lambda exonuclease selectively digests the 5'of the double-stranded DNA. Phosphorylated chain.
  • the amplified product was treated with Lambda exonuclease into single strands and purified by ultrafiltration.
  • Step 2 After PCR amplification, the PCR solution is ultrafiltered to remove salt, and then Lambda exonuclease is added. After a constant temperature reaction at 37°C for a certain period of time, the Lambda exonuclease is heat-inactivated, and then the digested product solution is again Purification by ultrafiltration.
  • the above step 3) mix the single-stranded DNA obtained in step 2) with the thio-DNA strand and RNA closed strand synthesized in step 1) in DNase I buffer solution, add DNase I after heating and annealing, and react at 37-42°C 30 ⁇ 45min, then heat inactivate DNase I.
  • the conditions for heating and annealing are melting at 95°C for more than 30s, then slowly lowering to room temperature, and the cooling rate does not exceed 10°C/min to prevent incomplete hybridization.
  • the specific procedure can be: 95°C for 90s; 80°C for 90s; 65°C for 90s; 50°C for 90s; 37°C for 120s.
  • the above step 4) methods for detecting DNase I digestion products include but are not limited to: Sanger sequencing detection after PCR amplification, pyrosequencing, next-generation sequencing, etc.
  • the thioDNA strands and closed RNA strands used in the present invention are both obtained by chemical synthesis, wherein the thioDNA strands will not be hydrolyzed by nuclease, and the RNA closed strands will not be cleaved by DNase I, and are separated from DNA in non-target regions. After strand hybridization, it can help inhibit DNase I from cleaving the DNA in the hybrid strand.
  • the two current mainstream gene mutation detection technologies are second-generation sequencing and digital droplet PCR.
  • the problem with the second-generation sequencing method is that the detection cycle is long, especially for low-abundance genetic mutation samples, which requires longer sequencing and data processing, usually 2-4 weeks, which is not only costly, but also difficult to meet the clinical needs of a large number of samples. The need for rapid testing.
  • the digital droplet PCR method relies on a large sample volume to provide sufficiently high sensitivity, which is unacceptable for patients.
  • the present invention removes wild-type DNA through one-step pretreatment, so that the mutation abundance rises to the range that can be measured by the Sanger sequencing method.
  • Figure 1 is a schematic diagram of the method of direct Sanger sequencing after the enrichment of low-abundance gene mutation samples by direct Sanger sequencing after the enrichment of low-abundance gene mutation samples by thiol DNA chain guiding DNase I to cut wild-type DNA to significantly increase the abundance of mutant DNA.
  • Figure 2 is a comparative diagram of the relative rates of DNase I digestion by EGFR L858R wild-type probe and mutant probe in Example 1 after hybridizing with different types of nucleic acid strands.
  • Figure 3 is a fluorescence curve diagram of the enzyme digestion reaction of EGFR L858R wild-type probe (a) and mutant probe (b) by DNase I under the guidance of different concentrations of thioDNA strands in Example 1, where in (b) The inset is an enlarged view of the four rising fluorescence curves.
  • Fig. 4 is a gel electrophoresis diagram of the products of EGFR L858R wild-type long-chain DNA and mutant long-chain DNA after RNA chain blocking by thio-DNA strand-guided DNase I in Example 1 at different temperatures for 30 minutes.
  • Fig. 5 is a gel electrophoresis diagram of the product of KRAS G13D wild-type long-chain DNA and mutant long-chain DNA after being digested with RNA strands blocked by DNase I guided by three different lengths of thioDNA strands in Example 1 for 30 minutes .
  • Fig. 6 is a Sanger sequencing chart of PCR products of samples with EGFR L858R mutation abundances of 0.1% and 0.01% in Example 2 after being pretreated with thioDNA-guided DNase I for different times.
  • Figure 7 shows the PCR products of standard and negative tissue samples with EGFR L858R mutation abundances of 1.0%, 0.1%, and 0.01%, positive tissue samples from lung cancer patients, and plasma samples in Example 2 before and after pretreatment with thioDNA-guided DNase I Sanger sequencing map.
  • Example 1 The thio-DNA chain guides DNase I to selectively remove the wild-type DNA chain>
  • two different target gene DNA sequences were selected, one is the sequence of the point mutation (L858R) at codon 858 in exon 21 of human epidermal growth factor receptor (EGFR); the other is the sequence of V -Ki-ras2Kirsten rat sarcoma virus oncogene (KRAS) the sequence of the point mutation (G13D) at codon 13 in exon 2 of Kirsten.
  • L858R the sequence of the point mutation at codon 858 in exon 21 of human epidermal growth factor receptor
  • KRAS V -Ki-ras2Kirsten rat sarcoma virus oncogene
  • EGFR L858R thioDNA chain guides DNase I to selectively cut EGFR L858R wild-type DNA chain
  • the sequences of EGFR L858R wild-type long-chain DNA and EGFR L858R mutant long-chain DNA are shown in Table 1.
  • the underlined part is the target region, and the mutation site is shown in bold.
  • the corresponding EGFR L858R thioDNA chain sequence is designed as: 5'-AAAAAAAAAAA GGGCTGGCCAA CGCAGATA-3', wherein the phosphate backbone is fully thio modified, and the underlined part is 11-nt which is completely complementary to the wild-type target sequence
  • the rest of the sequence is to increase the binding force to DNase I.
  • Both of the above two probes are labeled with a fluorescent group and a quenching group, and high-efficiency fluorescence resonance energy transfer can occur between the two.
  • the sequences of the two probes remain intact, the fluorescence emitted by the fluorophore after being excited is absorbed by the quenching group, so the fluorescence cannot be detected.
  • the quenching group is far away from the fluorescent group, and the solution will emit strong fluorescence, which can effectively indicate whether the probe sequence is selectively cleaved and cleaved by DNase I guided by thioDNA The speed and progress of the reaction.
  • step 2 2) Add 0.05 U DNase I to the annealed solution in step 1, and immediately put it into a real-time fluorescent PCR machine (Rotor Gene Q) after mixing, and measure the fluorescence value at 37°C.
  • the excitation wavelength is 470 ⁇ 10nm, and the detection wavelength It is 510 ⁇ 5nm.
  • the cleavage rate of the double-strand formed by the mutant probe is even slightly faster than that of the fully complementary double-strand formed by the wild-type probe; after adding the thio DNA strand in the fourth set of experiments, the wild-type probe and its fully complementary paired to form DNA: sulfur
  • the hybridized strands of the new generation DNA are quickly cleaved by DNase I, while the mutant probes are cleaved by DNase I at a rate slightly less than that of the mutant probes due to single-base mismatches with the thio DNA strands.
  • the mutant probes are not easy to directly bind to DNase I, nor are they easy to pass Hybridizes with the thio DNA strand and is cut, so it is retained instead.
  • the wild-type probes and mutant probes used in the above-mentioned fluorescence analysis experiments are both 16 nt short-stranded DNA, and in the actual genomic sample system, the test DNA is usually long-stranded.
  • the test DNA is usually long-stranded.
  • the sequence length is 130 nt, of which the length of the target region complementary to the thio DNA is 11 nt, and the rest are non-target regions.
  • the specific experimental steps are as follows:
  • EGFR L858R wild-type long-chain DNA or mutant long-chain DNA (20pmol), EGFR L858R RNA closed chain 1-4 (20pmol each), EGFR L858R thio DNA chain (25pmol) in 50 ⁇ L buffer solution, buffer The composition of the liquid is: 10mmol/L Tris-HCl, 2.5mmol/L MgCl 2 , 0.5mmol/L CaCl 2 , pH 7.6@25°C, annealing at elevated temperature.
  • lane 1 wild-type DNA long chain is added with RNA closed strand and thio-DNA strand, and DNase I is not added after heating and annealing
  • lane 2-5 wild-type DNA long Add RNA closed strands and thio-DNA strands to the strands. After heating and annealing, they are treated with DNase I for 30min. The reaction temperature is 33.3°C, 37.1°C, 41.7°C and 45.9°C.
  • Lane 6 Mutant DNA long strands add RNA closed strands and sulfur DNA strands, no DNase I treatment after heating and annealing; Lane 7-10: Mutant DNA long strands are added to the closed RNA strands and thio DNA strands, and after heating and annealing, they are treated with DNase I for 30 minutes. The reaction temperature is 33.3°C, 37.1 °C, 41.7°C and 45.9°C. Observe the distribution of DNA bands after staining with GelRed nucleic acid dye.
  • KRAS G13D thioDNA chain guides DNase I to selectively cut KRAS G13D wild-type DNA chain
  • KRAS G13D wild-type long-chain DNA or KRAS G13D mutant long-chain DNA (20pmol), KRAS G13D RNA closed strand 1-4 (20pmol each), and KRAS G13D thioDNA strand A/B/C (25pmol).
  • the composition of the buffer is: 10mmol/L Tris-HCl, 2.5mmol/L MgCl 2 , 0.5mmol/L CaCl 2 , pH 7.6@25°C, heating annealing.
  • Lanes 1, 3, 5, and 7 correspond to the detection results of wild-type long-chain DNA, and lanes 2, 4, 6, and 8 correspond to the detection results of mutant long-chain DNA.
  • Lane 1-2 Without adding DNase I; Lane 3-4: Adding thioDNA strand A and DNase I for 30 minutes; Lane 5-6: Adding thio DNA strand B and DNase I for 30 minutes; Lane 7-8: Adding Treat the thioDNA strand C and DNase I for 30 minutes. The experimental results are shown in Figure 5.
  • the sequences of the wild-type DNA long chain, mutant DNA long chain, thio DNA chain and RNA closed chain of EGFR L858R used in this embodiment are listed in Table 1.
  • the sequence of PCR amplification primers used is:
  • EGFR L858R forward primer 5'-TTCTTTCTCTTCCGCACC-3' (5'-OH end) (SEQ ID No: 21)
  • EGFR L858R phosphorylation reverse primer 5'-PO 4 -TACTTGGAGGACCGTCG-3'(5' end phosphorylation tag) (SEQ ID No: 22)
  • Enzyme digestion of genomic DNA with Shearase enzyme Mix 1 mg of genomic DNA with 1.5 ⁇ L Shearase enzyme in 20 ⁇ L buffer, the buffer composition is 10mmol/L Tris-HCl, 25mmol/L MgCl 2 , 1mmol/L DTT, pH 7.5@25°C, incubate at 42°C for 15min, then heat-inactivate the Shearase enzyme.
  • PCR amplification of genomic DNA and standard DNA 1.5 ⁇ L of the product solution in step 1) or 0.25amol DNA standard, 20pmol forward primer, 20pmol reverse primer, 1nmol dNTPs, 1 ⁇ L LC Green, 0.5U Q5 polymerase was mixed thoroughly in 50 ⁇ L Q5 buffer for PCR amplification (program: 98°C 60s; 98°C 9s, 63.5°C 18s, 72°C 20s, cycle 35 cycles; 72°C extension 600s).
  • thioDNA strand to guide DNase I to selectively remove the wild-type DNA strand: combine the genomic DNA or standard DNA (1pmol) obtained in step 3), the closed RNA strand (10pmol each) and the thioDNA strand (12.5pmol) ) Mixed in 50 ⁇ L buffer solution, the buffer composition is: 10mmol/L Tris-HCl, 2.5mmol/L MgCl 2 , 0.5mmol/L CaCl 2 , pH 7.6@25°C, add 0.05U DNase to the solution after heating and annealing I, react at 37°C for 30 min, then heat inactivate DNase I.
  • PCR amplification of DNase I digestion product 1.5 ⁇ L of the DNase I digestion product solution obtained in step 4), 20pmol forward primer, 20pmol reverse primer, 1nmol dNTPs, 1 ⁇ L LC Green, 0.5U Q5 polymerization Enzymes were thoroughly mixed in 50 ⁇ L Q5 buffer for PCR amplification (program: 98°C 60s; 98°C 9s, 63.5°C 18s, 72°C 20s, cycle 35 cycles; 72°C extension 600s).
  • step 4 was pretreated for different times (0 min, 18 min, 45 min, 90 min), and then step 5) PCR amplification and step 6) Sanger sequencing were performed.
  • the resulting Sanger sequencing pattern was as shown in 6 Shown. Sanger sequencing could not detect the presence of mutation peaks at the target site for samples (0 min) that were not digested with DNase I guided by thioDNA. After processing the sample, the signal of the target site mutation peak can be detected in the Sanger sequencing map.
  • the signal of the mutation peak in the Sanger map gradually increases, indicating that the abundance of the mutant chain in the system to be tested is significantly increased with the selective digestion of the wild chain by DNase I
  • the improvement also confirmed that the RNA block chain effectively blocks the non-target area of the substrate chain.
  • the initial mutation abundance of 1%, 0.1%, and 0.01% of the samples in the Sanger map have mutation peak signals of about 60%, 30% and respectively. 10%.
  • gene mutation sites can also be detected by Sanger sequencing.
  • the mutation abundance in the Sanger profile of lung cancer-positive tissue samples was also significantly increased after processing. After the negative tissue samples were processed, there was no false positive signal in the Sanger profile.
  • the method of the present invention makes the low-abundance gene mutation sample that cannot be directly detected by the Sanger sequencing method after selective enzyme digestion treatment can increase the abundance of the mutant chain to be directly detected by the Sanger sequencing method.
  • the processing process only takes 4 hours. From getting the clinical sample to giving the Sanger sequencing result, the entire analysis process takes no more than 24 hours. This provides a powerful means for rapid sequencing analysis of clinical samples and has a good prospect for promotion and application.

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Abstract

一种基于选择性消除野生链背景干扰的基因突变检测方法，包括：通过PCR对待测目标序列进行扩增，并通过Lambda核酸外切酶处理成单链DNA；设计并合成与野生型DNA序列目标区域互补的硫代DNA链以及与非目标区域互补的RNA封闭链，与单链DNA混合并升温退火后加入DNase I进行切割；DNase I在硫代DNA链的引导下选择性地切除野生型DNA链目标区域序列，突变型DNA链由于目标区域内存在错配不能被DNase I切除而得以保留，从而显著提升突变型DNA链的丰度，大幅度降低检测低丰度基因突变的难度。本发明方法无需复杂昂贵的仪器，容易操作，成本低，1天之内即可给出结果，可为临床肿瘤早筛和术后复发监测提供及时、可靠的基因突变信息。

Description

一种基于选择性消除野生链背景干扰的基因突变检测方法 技术领域
本发明涉及基因组样品处理和基因突变检测领域，特别涉及一种对基因组样品进行预处理的方法，通过该方法消除野生型DNA后，与常规测序法联用，实现超低丰度基因突变的快速、低成本测序分析。
背景技术
癌症(恶性肿瘤)是当今严重威胁人类健康的头号杀手，与癌症相关的早期诊断和术后复发监测具有重要的生物学和医学意义。基因突变是指基因组DNA分子发生了可遗传的变异现象，是导致恶性肿瘤形成的重要原因之一。传统的肿瘤基因检测手段主要是组织活检，即通过手术穿刺的方式获取肿瘤组织样本并对其进行基因检测，该方法主要针对癌变组织进行检测，对于还未形成病灶的早期肿瘤细胞无法实现有效的检测，对于已经形成病灶的肿瘤组织也可能因为肿瘤异质性而得到假阴性的检测结果。此外，这种侵入式的检测手段给患者带来较大的痛苦，且可能在手术穿刺时进一步刺激癌组织引发恶化。组织活检也不适合进行连续取样和病情跟踪检测。
随着精准医疗概念的提出和循环肿瘤DNA(Circulating tumor DNA,ctDNA) 1、循环肿瘤细胞和外泌体等新型肿瘤标志物的发现，液体活检逐渐成为癌症早期诊断的新希望。ctDNA是早期肿瘤细胞脱落或者凋亡后释放至循环***内的DNA，该DNA高度碎片化但携带肿瘤细胞的全部基因突变信息。针对循环体系内ctDNA的检测能够为肿瘤早筛和复发监测提供重要的信息。由于正常细胞在程序性凋亡的过程中也会向循环***中释放大量的碎片化DNA，给血浆游离DNA(Cell free DNA，cfDNA)中ctDNA的检测带来很大程度的背景干扰。据文献报道，血浆中存在基因突变的ctDNA丰度一般在0.001％-10％之间，这对突变检测方法的灵敏度和选择性都提出了很高的要求。
目前针对基因突变的检测方法主要有测序法、数字液滴PCR法和DNA探针杂交法等。直接测序法是最为经典、适用范围最广的基因突变检测方法。测序法能够直接给出目标DNA分子的核苷酸序列，因此被视作基因突变检测的金标准。Sanger测序法(双脱氧链终止法)经过数十年的发展和技术优化，实现了单一样品的低成本快速检测，但其灵敏度有限，对突变基因丰度的最低检测限在5％-10％之间，无法直接用于ctDNA的检测。第二代测序法(Next-generation sequencing，NGS) 2采用一种与经典的Sanger链终止法截然不同的原理， 它以大规模并行的方式，实现边合成边测序(Sequencing by synthesis，SBS)。在常规测序深度下，NGS能够给出有效数据的突变丰度范围为0.1-0.5％以上。在此基础上，Newman等人开发了肿瘤个体化深度测序分析法(Cancer personalized profiling by deep sequencing，CAPP-NGS)。该方法的核心思想是缩小NGS的检测范围，通过对人群大样本的分析，筛选出约0.04％目标范围的基因库，仅对该基因库进行高深度二代测序，检测限可以达到0.02％，并同时实现数百个基因突变的同时检测。但是CAPP-NGS仍然不能够满足早期癌症样品的检测，且为了达到0.02％的检测限，CAPP-NGS的测序深度需10000倍以上，检测成本极高，检测周期达数周，很难大范围推广。在二代测序被开发和商业化的同时，以纳米孔(Nanopore) 3测序为代表的第三代测序技术同样得到了广泛的关注。Nanopore测序利用α-溶血素构建生物纳米孔，孔中间恰好允许单链DNA通过。DNA链通过纳米孔时会阻碍其他离子自由进出纳米孔从而导致纳米孔附近的电流发生变化，此外，DNA上四种碱基的化学结构和大小略有不同，对纳米孔附近电流的实时监测可以间接获取到DNA的序列信息。Nanopore技术最大的特点是惊人的读长以及小巧的体积，在实际应用中最高达到了147Kb。但目前测序结果的准确度还不够高，仅有80％，与实现ctDNA的直接测序还有一定的距离。
数字液滴PCR(digital droplet PCR,ddPCR) 4在传统的PCR技术的基础上，利用芯片或者液滴实现样品的独立分配，对独立液滴进行PCR，极限检测限能达到0.005-0.05％，是目前检测ctDNA灵敏度最高的方法，但达到该极限检测限对于原PCR体系超分散至微滴时的要求极高。为了保证测定的准确性，该方法稳定的检测限被人为控制在0.01％-0.1％之间，实际临床使用时将检测限控制在0.05％-0.1％。检测限越低，所需的样品量就越大。ddPCR关键的技术核心为实现待测DNA单分子级别的分散，实际操作繁琐，仪器价格昂贵，操作成本高，检测周期2-4天，也不易实现ctDNA的快速分析。
荧光探针法利用人为设计并合成的分别标记有荧光基团和猝灭基团的单链DNA探针，特异性识别并结合体系中的待测突变目标链并给出荧光信号，从而实现待测样品中突变基因丰度的检测。在一般测定条件下，待测样品中野生链与突变链间的差异仅为一个碱基，DNA探针对于该碱基的区分能力有限，在实际应用过程中检测限为10％左右。为了进一步提高DNA荧光探针的灵敏度，人们在简单探针的基础上发展了多种新型探针，如分子信标、二元探针、三茎探针等，将检测限降低至3％。Zhang 5等人进一步在简单的DNA杂交探针的基础上引入大量热力学和动力学计算，利用链竞争、链置换、链迁移的识别进一步将检测限降低至0.01％至1％，但是该方法检测时间较长且需要对于探针序列和竞争序列进行精确的计算。 Das 6等人结合DNA探针与高灵敏度的电化学检测法开发出了一种基于探针杂交免PCR的基因突变电化学检测法，该方法借助电化学检测的高灵敏度，摒弃传统PCR，能够直接测定0.01％的突变链，达到了超低丰度基因突变检测水平。但该方法的普适性较差，纳米电极的构造以及电极表面纳米载体的合成较为困难，针对不同的检测体系需要重新设计电化学检测体系，同时体系中大量使用的PNA成本较高，限制了该方法的进一步推广。Xiao 7等人在原有荧光探针法的基础上引入了核酸酶辅助荧光信号放大显著提高了荧光探针法的灵敏度。相比于测序法，荧光分析法缺少碱基变化的直接信息。
目前针对实际样品的检测还离不开PCR扩增，通过提高PCR过程的选择性对样品体系中突变链进行优先富集的方法也得到了广泛的关注和研究。将扩增阶段的常规PCR替换成选择性PCR可以在最终测定前提高待测样品中突变链的丰度，从而提高对低丰度突变的灵敏度。现有的选择性PCR方法主要包括突变阻滞扩增PCR(Amplification refractory mutation system PCR，ARMS PCR) 8、野生链阻碍PCR(Wild-type blocking PCR) 9、低温PCR(Co-amplification at lower temperature PCR，COLD-PCR) 10、锁核酸/肽核酸钳PCR(Locked nucleic acids/peptide nucleic acids-mediated PCR，LNA/PNA-mediated PCR) 11以及将上述方法联用的ARMS-qPCR、ice-COLD PCR等方法。但是PCR体系本身就是一个涉及多温度组合、多组分参与的复杂生化反应，在引物设计、温度优化和时间控制上都较为繁琐，产物组成也十分复杂。在PCR过程中额外引入竞争性的新组分，使反应变得更加复杂难控。在实验过程中需要对各核酸链的序列、各组分加入量、退火温度及各阶段恒温时间进行复杂的优化和精确的控制，影响因素复杂。即使是组合型选择性PCR，其稳定的检出限也控制在0.05％-0.1％之间，难于满足ctDNA的快速测序分析要求。
由于野生型DNA不存在突变，不携带关键致病基因相关信息，亦不能为肿瘤早筛或者术后复发监测提供关键信息，却在检测体系中占据绝大多数比例，给样品中突变型DNA的检出带来巨大的背景干扰。如果能构建一种简单、高效的能选择性去除体系中野生型DNA的方法，则可以显著提升突变型DNA的丰度，之后可通过常规测序方法进行分析。在这方面目前有两个研究组开展过一些探索性研究，分别是利用双链特异性核酸酶(DSN酶) 12以及磁珠捕获(DISSECT) 13的方法选择性去除待测样品中大量的野生型DNA。但两种方法均存在明显的局限性，难以获得稳定、可靠的结果。例如，DSN酶本身对于野生型DNA和突变型DNA并没有序列选择性，其区分能力仅来源于底物链是单链还是双链，辅之以精确的温度控制。这一原理导致基因组的长链DNA可能会被非特异性水解，从而导致富集的失败。 另一项DISSECT技术是利用互补序列修饰的磁珠选择性杂交去除体系中的野生型DNA。由于该去除过程是线性的，单次去除效率较低，而且磁珠表面的非特异性吸附易导致突变型DNA链的损失。
我们课题组在以前的研究中曾通过DNase I和硫代DNA链在高浓度下的静电自组装作用构建了一种可调控核酸酶序列选择性的酶复合物(专利号：ZL 2014 1 0797398.6) 14。该酶复合物可以高效率地选择性切除体系中与硫代DNA链完全互补配对的底物链，这为我们开发一种选择性去除基因组样品中野生链的方法奠定了重要的基础。
综上所述，现有的方法在针对ctDNA的早期肿瘤检测方面仍然存在灵敏度不够高，检测周期长，方法不稳定以及实验条件控制要求苛刻和仪器试剂价格高昂等一系列问题。针对这些问题，本发明拟发展一种灵敏度高、操作相对简单、结果稳定性好、成本较低、易于推广使用和服务于临床检验的基因突变检测方法。
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发明内容
本发明的目的在于提供一种基因突变检测方法，通过构建一种简便、高效的方法去除待测体系中的野生型DNA，从而使突变链DNA的丰度上升到可以直接用Sanger测序法检出。
我们对专利ZL 2014 1 0797398.6所建立的方法开展深入机理研究的过程中发现，DNase I与硫代DNA链之间的亲和力明显大于该酶与天然DNA单链之间的亲和力。这意味着在一个混合体系中，DNase I会优先与硫代DNA链结合，然后才与普通DNA链相互作用。利用这一特点，本发明构建了一个无需预组装，在低浓度DNase I的条件下，由溶液中游离的硫代DNA链优先结合DNase I，然后在溶液中原位诱导DNase I选择性切割与硫代DNA链完全互补序列的方法。根据待测目标基因的DNA序列，我们将硫代DNA链的序列设计成与野生型DNA完全互补，使之被快速水解；而突变链由于与硫代DNA链存在单碱基错配，基本不被切割。经过这一体系处理过的基因组样品，突变链所占的比例显著提高，后续直接用最简单的Sanger测序法即测出基因组样品中是否存在突变和突变碱基的类型。进一步通过处理时间与突变富集倍数之间的定量关系，可计算出原始基因组样品中的突变丰度值。
本发明方法的原理如图1所示，将突变型DNA链与野生型DNA链序列中可能存在突变 位点的区域(10-12个碱基范围)记为目标区域，人为设计硫代DNA链序列与野生型DNA链目标区域序列完全互补配对，突变型DNA链则由于突变位点的存在其目标区域不能与硫代DNA链序列完全互补配对。突变型DNA链与野生型DNA链目标区域外的序列记为非目标区域，二者在非目标区域内序列一致。为了防止非目标区域被DNase I非特异性切割，在溶液中加入与其序列完全互补的RNA单链对其进行封闭。
将上述方法应用于分析基因组样品(如血浆样品或者组织样品)时，首先利用试剂盒提取和纯化其中的DNA，然后通过PCR对所提取DNA中待测目标序列(野生型或突变型)进行扩增。扩增产物经Lambda核酸外切酶处理成单链DNA后，其中含有大量的野生型DNA单链和少量的突变型DNA单链。向体系中加入RNA封闭链和硫代DNA链，升温退火后加入DNase I进行切割。DNase I在硫代DNA的引导下选择性地切除野生型DNA链目标区域序列，而突变型DNA链由于目标区域内存在错配不能被DNase I切除而得以保留在溶液体系中。随着酶切反应的进行，突变型DNA链的丰度显著提升，直到常规突变检测方法可准确检出的范围，后续通过PCR扩增和Sanger测序即可直接检测。
本发明的基因突变检测方法具体操作步骤如下：
1)确定基因组DNA待测目标序列，该目标序列包括目标区域和非目标区域，设计并合成与野生型DNA序列目标区域互补的硫代DNA链以及与非目标区域互补的RNA封闭链；
2)设计PCR扩增体系对待测目标序列(包括野生型和突变型)进行扩增，利用Lambda核酸外切酶将扩增产物处理成单链；
3)将步骤2)得到的单链DNA与步骤1)合成的硫代DNA链和RNA封闭链在DNase I缓冲溶液中混合，升温退火后加入DNase I反应一段时间，热失活DNase I；
4)对步骤3)酶切产物进行测序检测。
上述步骤1)中，所述待测目标序列的长度优选为115～130nt，可能存在突变位点的目标区域优选位于待测目标序列的中部。所述目标区域中可能存在一个或多个突变位点，根据序列GC含量不同，目标区域长度控制在其熔解温度Tm在42-46℃之间，一般的，目标区域的长度优选为11～13nt。
所述非目标区域位于目标区域的两端，与非目标区域互补的RNA封闭链的长度通常为25～90nt，如果非目标区域比较长，可以设计并合成多条RNA封闭链。
所述硫代DNA链的长度优选为16～59nt，包括与野生型DNA目标区域互补片段和两端的非互补片段。优选的，所述硫代DNA链为全硫代DNA链。
上述步骤2)中，优选的，PCR扩增体系的正向引物为5’-OH末端，反向引物的5’末端磷酸化标记，Lambda核酸外切酶选择性地消化双链DNA的5’磷酸化的链。扩增产物经Lambda核酸外切酶处理成单链后通过超滤纯化。
步骤2)中PCR扩增后，先对PCR溶液进行超滤除盐，然后加入Lambda核酸外切酶，37℃恒温反应一定时间后热失活Lambda核酸外切酶，随后对酶切产物溶液再次进行超滤纯化。
上述步骤3)将步骤2)得到的单链DNA与步骤1)合成的硫代DNA链和RNA封闭链在DNase I缓冲溶液中混合，升温退火后加入DNase I，在37～42℃温度下反应30～45min，然后热失活DNase I。其中升温退火的条件是95℃下30s以上解链，然后缓慢降至室温，降温速率不超过10℃/min，防止杂交不完全。具体程序可以是：95℃ 90s；80℃ 90s；65℃ 90s；50℃ 90s；37℃ 120s。上述步骤4)对DNase I酶切产物检测的方法包括但不限于：PCR扩增后Sanger测序检测、焦磷酸测序、二代测序等。
本发明中使用的硫代DNA链和RNA封闭链均通过化学合成法获得，其中硫代DNA链不会被核酸酶水解，RNA封闭链本身不会被DNase I切割，与非目标区域的DNA单链杂交后可以辅助抑制DNase I对于杂交链中DNA的酶切。
与现有技术相比，本发明具有明显的技术优势，具体阐述如下：
现行的两类主流的基因突变检测技术是二代测序和数字液滴PCR。二代测序法面临的问题是检测周期长，尤其是对低丰度的基因突变样品，需要更长时间的测序和数据处理，通常是2-4周，不仅成本高，而且难以满足临床大量样品快速检测的需求。数字液滴PCR法则依赖于较大的样品量，才能提供足够高的灵敏度，这对患者来说是不易接受的。本发明通过一步预处理去除野生型DNA，使突变丰度上升到Sanger测序法可以测到的范围。实验证实，这一方法对突变丰度低至0.01％的样品仍可以取得明显的富集效果，这大幅度降低了目前已有技术检测低丰度基因突变的难度。整个方法无需复杂昂贵的仪器，容易操作，成本低，1天之内即可给出结果，比二代测序法更具有实际应用价值，可为临床肿瘤早筛和术后复发监测提供及时、可靠的基因突变信息。
附图说明
图1是硫代DNA链引导DNase I切割野生型DNA以显著提高突变型DNA丰度，从而实现低丰度基因突变样品富集后直接Sanger测序的方法原理图。
图2是实施例1中EGFR L858R野生型探针和突变型探针与不同种类核酸链杂交后被 DNase I酶切的相对速率对比图。
图3是实施例1中DNase I在不同浓度硫代DNA链引导下对EGFR L858R野生型探针(a)和突变型探针(b)的酶切反应荧光曲线图，其中(b)中的内插图为四条荧光上升曲线的放大图。
图4是实施例1中在不同温度下，硫代DNA链引导DNase I对经过RNA链封闭后的EGFR L858R野生型长链DNA和突变型长链DNA酶切30min后产物的凝胶电泳图。
图5是实施例1中由三种不同长度的硫代DNA链引导DNase I对经过RNA链封闭后的KRAS G13D野生型长链DNA和突变型长链DNA酶切30min后产物的凝胶电泳图。
图6是实施例2中EGFR L858R突变丰度为0.1％和0.01％的样品经过硫代DNA引导DNase I预处理不同时间后PCR产物的Sanger测序图谱。
图7是实施例2中EGFR L858R突变丰度为1.0％、0.1％和0.01％的标准品和阴性组织样品、肺癌患者阳性组织样品及血浆样品经过硫代DNA引导DNase I预处理前后PCR产物的Sanger测序图谱。
具体实施方式
下面结合附图，通过具体实施例进一步阐述本发明。本领域的技术人员应当理解这些实施例仅用于说明本发明而不限制本发明的范围。
实施例1<硫代DNA链引导DNase I选择性切除野生型DNA链>
在该实施例中，选择了两种不同的目标基因DNA序列，一是人表皮生长因子受体(EGFR)21号外显子858号密码子处的点突变(L858R)所在序列；另一是V-Ki-ras2Kirsten大鼠肉瘤病毒癌基因(KRAS)2号外显子13号密码子处的点突变(G13D)所在序列。
表1.在本实施例中所用核酸链的序列(5’-3’)
Figure PCTCN2019113859-appb-000001
Figure PCTCN2019113859-appb-000002
Figure PCTCN2019113859-appb-000003
(1)EGFR L858R硫代DNA链引导DNase I选择性地切割EGFR L858R野生型DNA链
EGFR L858R野生型长链DNA和EGFRL858R突变型长链DNA的序列如表1中所示，下划线部分为目标区域，其中的突变位点加粗表示。针对该目标区域，设计对应的EGFR L858R硫代DNA链序列为：5’-AAAAAAAAAAA GGGCTGGCCAACGCAGATA-3’，其中的磷酸骨架全硫代修饰，下划线部分为与野生型目标序列完全互补的11-nt序列，其余部分是为了增加与DNase I的结合力。
为了更直观地展示该硫代DNA序列对野生链和突变链切割速率的差异，我们分别合成了用荧光标记的、长度为16nt的野生型和突变型短目标序列，分别称为EGFR L858R野生型探针和EGFR L858R突变型探针(序列见表1)，其中EGFR L858R野生型探针与EGFR L858R硫代DNA链存在11nt序列完全互补配对，EGFR L858R突变型探针与EGFR L858R硫代DNA链在对应的11nt序列中有一对碱基错配。
以上两种探针均标记了荧光基团和猝灭基团，二者之间可以发生高效率的荧光共振能量转移。当两种探针序列保持完整时，荧光基团受激后发出的荧光被猝灭基团吸收，因而检测不到荧光。而当这些序列被DNase I切割后，猝灭基团远离荧光基团，溶液将发出强烈的荧光，从而可以有效地指示该探针序列是否被硫代DNA引导的DNase I选择性地切割以及切割反应的快慢和进行程度。
具体实施步骤如下：
1)分别将20pmol野生型探针或突变型探针与20pmol硫代DNA链混合于50μL缓冲溶液(组成为:10mmol/L Tris-HCl,2.5mmol/L MgCl 2,0.5mmol/L CaCl 2,pH 7.6@25℃)中，然后将溶液升温退火(具体程序为95℃ 90s；80℃ 90s；65℃ 90s；50℃ 90s；37℃ 120s)。
2)向步骤1)退火后的溶液加入0.05U DNase I，混合均匀后立刻放入实时荧光PCR仪(Rotor Gene Q)中，在37℃下测定荧光值，激发波长为470±10nm，检测波长为510±5nm。
用野生型和突变型探针按照以上步骤共进行了4组对照实验，其中第一组不加硫代DNA链；第二组将硫代DNA链替换成与野生型探针完全互补配对的EGFR L858R RNA互补链(序列见表1)；第三组将硫代DNA链替换成与野生型探针完全互补配对的EGFR L858R DNA互补链(序列见表1)；第四组按正常步骤加入前述的与野生型探针完全互补配对的EGFR L858R硫代DNA链。四组实验得到的野生型探针和突变型探针被酶水解荧光上升的速率如图2所示。
可见，两种探针本身被DNase I切割的速率都很慢，且二者之间没有明显的差别。加入RNA互补链后，与两种探针均能形成DNA∶RNA杂交链，二者被DNase I切割的速率仍很慢且差别不大。第三组实验加入DNA互补链后，野生型探针形成完全互补配对的DNA双链，突变型探针形成单碱基错配的DNA双链，两种DNA双链均被DNase I快速水解，突变型探针形成的双链的切割速率甚至略快于野生型探针形成的完全互补的双链；第四组实验加入硫代DNA链后，野生型探针与其完全互补配对形成DNA∶硫代DNA杂交链，从而被DNase I快速切割，而突变型探针由于与硫代DNA链存在单碱基错配，被DNase I切割的速率甚至略小于突变型探针本身。这四组实验的对照结果充分证明了硫代DNA可引导DNase I选择性地切割与硫代DNA序列完全互补配对的野生型DNA链，而对仅存在单碱基错配的突变型DNA链基本没有影响。
为了进一步确证硫代DNA的引导作用，我们进一步设计了四组对照实验。每组实验中野生型探针和突变型探针的加入量均为20pmol，从第一组到第四组硫代DNA链的加入量依次为0、1pmol、10pmol和25pmol，对应硫代DNA链的终浓度分别为0、20nM、200nM和500nM，实时荧光分析的结果如图3所示。
由图3(a)的数据可以看到，随着硫代DNA链加入量的增加，野生型探针被DNase I切割的速率逐渐加快。当硫代DNA链的加入量大于野生型探针时，荧光信号迅速达到平台，表明野生型探针很快被DNase I水解清除掉了。而突变型探针则表现出相反的结果，如图3(b)所示，随着硫代DNA链加入量的增加，突变型探针被DNase I切割的速率越来越慢，进一步说明硫代DNA链与DNase I的结合力大于普通DNA单链与DNase I的结合力，当硫代DNA链与DNase I优先结合后，突变型探针既不容易直接与DNase I结合，也不容易通过 与硫代DNA链杂交而被切割，因此反而被保留下来。
在上述荧光分析实验中使用的野生型探针和突变型探针均为16nt的短链DNA，而在实际的基因组样品体系中，待测DNA通常为长链。为了防止长链DNA单链中目标区域两侧的序列因自身形成二级结构或链间产生部分杂交而导致被DNase I非特异性地切割，我们参照图2中第二组实验的结果，通过向溶液中加入非目标区域的RNA互补链对其进行保护。EGFR L858R野生型和突变型长链DNA的序列如表1所示，序列长度为130nt，其中与硫代DNA互补的目标区域长度为11nt，其余部分均为非目标区域。根据目前可大量合成RNA的长度限制，我们设计了4条长度在25nt–30nt之间的RNA封闭链(序列见表1)用于封闭长链DNA序列中非目标区域的部分。由于长链DNA序列上未标记荧光基团，我们采用凝胶电泳法表征了在硫代DNA链引导下、RNA链封闭的条件下，DNase I切割长链DNA的产物情况。具体实验步骤如下：
1)将EGFR L858R野生型长链DNA或突变型长链DNA(20pmol)，EGFR L858R RNA封闭链1-4(各20pmol)，EGFR L858R硫代DNA链(25pmol)混合于50μL缓冲溶液中，缓冲液组成为：10mmol/L Tris-HCl,2.5mmol/L MgCl 2,0.5mmol/L CaCl 2,pH 7.6@25℃，升温退火。
2)对野生型长链DNA和突变型长链DNA均设计不加DNase I、加入0.05U DNase I和加酶后不同温度(33.3℃至45.9℃之间)下反应的对照实验，反应时间均为30min，之后加热使DNase I失活。取少量产物溶液(9μL)进行琼脂糖凝胶电泳实验。
3)设计十组2.5％琼脂糖凝胶电泳实验，其中泳道1：野生型DNA长链加入RNA封闭链和硫代DNA链，升温退火后不加DNase I；泳道2-5：野生型DNA长链加入RNA封闭链和硫代DNA链，升温退火后经DNase I处理30min，反应温度依次为33.3℃、37.1℃、41.7℃和45.9℃；泳道6：突变型DNA长链加入RNA封闭链和硫代DNA链，升温退火后不加DNase I处理；泳道7-10：突变型DNA长链加入RNA封闭链和硫代DNA链，升温退火后经DNase I处理30min，反应温度依次为33.3℃、37.1℃、41.7℃和45.9℃。用GelRed核酸染料进行染色后观察DNA条带分布。
上述凝胶电泳的结果如图4所示。对比泳道1和泳道6可见，在不加入DNase I的情况下，野生型DNA长链和突变型DNA长链均与4条RNA封闭链结合形成了杂交双链，长度为130bp。对比泳道1-5可知，在四种不同的反应温度下，野生型DNA长链与RNA封闭链形成的杂交底物链均可被DNase I快速酶切，酶切产物长度约为80bp和30bp，分别对应于 底物链5’末端和3’末端目标区域外序列与RNA封闭链形成的杂交双链。该结果表明RNA封闭链的杂交有效防止了DNase I对于非目标区域的切割，且并不影响体系中的硫代DNA链引导DNase I对于目标区域高效率的切割。对比泳道6-10可知，突变型DNA长链与RNA封闭链形成的杂交底物链在33.3 o下经DNase I作用30min后，有少量被水解。随着温度的升高，杂交产物稳定性下降，越来越多地被DNase I水解。温度升高到45.9℃时，大部分杂交产物都被水解。与泳道1-5所示野生型DNA长链的产物条带比较，突变型DNA长链的水解产物并不集中为清晰的两个条带，说明水解过程并无选择性，是随机发生的。以上实验结果表明，突变型长链DNA的非目标区域被RNA链封闭保护后，目标区域仍可被硫代DNA链引导的DNase I选择地切割。
(2)KRAS G13D硫代DNA链引导DNase I选择性地切割KRAS G13D野生型DNA链
为了证明硫代DNA链可引导DNase I选择性切除野生型DNA长链目标区域序列这一性质的普适性，我们进一步选择KRAS G13D基因突变位点所在序列开展了研究。KRAS G13D野生型长链DNA，KRAS G13D突变型长链DNA以及针对两条链非目标区域进行封闭的KRAS G13D RNA封闭链1–4的序列如表1所示。此外，我们设计合成了三条不同长度的硫代DNA链进行对比，分别为KRAS G13D硫代DNA链A(41nt)、B(42nt)和C(43nt)，其与野生型DNA链目标区域互补配对的序列长度分别为10nt，11nt和12nt。
通过琼脂糖凝胶电泳实验对三条不同硫代DNA链引导DNase I选择性切除长链底物中目标区域序列的效果进行了比较，具体操作步骤如下：
1)将KRAS G13D野生型长链DNA或KRAS G13D突变型长链DNA(20pmol)，KRAS G13D RNA封闭链1-4(各20pmol)，KRAS G13D硫代DNA链A/B/C(25pmol)混合于50μL缓冲溶液中，缓冲液组成为：10mmol/L Tris-HCl,2.5mmol/L MgCl 2,0.5mmol/L CaCl 2,pH 7.6@25℃，升温退火。
2)根据实验设计，向实验组中加入0.05U DNase I，对照组中不加入DNase I，37℃下酶切30min，酶切结束后热失活DNase I。
3)利用2.5％琼脂糖凝胶电泳确定2)中产物溶液DNA条带分布。
共设计八组2.5％琼脂糖凝胶电泳实验，用GelRed核酸染料进行染色后观察。其中泳道1、3、5、7对应野生型长链DNA的检测结果，泳道2、4、6、8对应突变型长链DNA的检测结果。泳道1-2：不加DNase I；泳道3-4：加入硫代DNA链A和DNase I处理30min；泳道5-6：加入硫代DNA链B和DNase I处理30min；泳道7-8：加入硫代DNA链C和DNase I 处理30min。实验结果如图5所示。对比泳道1-8可见，野生型DNA长链和突变型DNA长链经过RNA封闭链封闭后，三条硫代DNA链均能够引导DNase I选择性切割野生型DNA长链目标区域序列。
以上研究结果表明，在实际应用过程中，硫代DNA链的序列可根据待处理底物链目标区域序列进行灵活设计，本发明的方法具有良好的序列普适性，设计和实施也都简便易行。
实施例2<通过切除野生型DNA链实现低丰度EGFR L858R基因突变的快速测序分析>
本实施例中所用EGFR L858R的野生型DNA长链、突变型DNA长链、硫代DNA链和RNA封闭链的序列均见表1所列。所用PCR扩增引物的序列为：
EGFR L858R正向引物：5’-TTCTTTCTCTTCCGCACC-3’(5’-OH末端)(SEQ ID No：21)
EGFR L858R磷酸化反向引物：5’-PO 4–TACTTGGAGGACCGTCG-3’(5’末端磷酸化标记)(SEQ ID No：22)
实验操作步骤如下：
1)用Shearase酶对基因组DNA进行酶切处理：将1mg基因组DNA与1.5μL Shearase酶充分混合于20μL缓冲液中，缓冲液组成为10mmol/L Tris-HCl，25mmol/L MgCl 2，1mmol/L DTT，pH 7.5@25℃，在42℃下孵育15min，随后热失活Shearase酶。
2)对基因组DNA和标准品DNA进行PCR扩增：将1.5μL步骤1)中的产物溶液或0.25amol DNA标准品，20pmol正向引物，20pmol反向引物，1nmol dNTPs，1μL LC Green，0.5U Q5聚合酶充分混合于50μL Q5缓冲液中进行PCR扩增(程序为：98℃ 60s；98℃ 9s,63.5℃ 18s,72℃ 20s,循环35圈；72℃延伸600s)。
3)PCR扩增产物的单链化和纯化：对步骤2)中溶液进行超滤除盐(超滤溶剂：去离子无酶水，超滤管截断分子量30KDa，超滤温度4℃，超滤时间：10min，转速：6000rpm，超滤次数：2次)。取出超滤管中上层溶液置于PCR管中(液体总体积约为50μL)，向其中加入5U Lambda核酸外切酶，37℃恒温反应10min后热失活Lambda核酸外切酶。随后对酶切产物溶液再次进行超滤纯化，超滤方法同上，用Qubit 3.0确定超滤后上层溶液中DNA的含量。
4)利用硫代DNA链引导DNase I选择性切除野生型DNA链：将步骤3)中得到的基因组DNA或者标准品DNA(1pmol)，RNA封闭链(各10pmol)和硫代DNA链(12.5pmol) 混合于50μL缓冲溶液中，缓冲液组成为：10mmol/L Tris-HCl,2.5mmol/L MgCl 2,0.5mmol/L CaCl 2,pH 7.6@25℃，升温退火后向溶液中加入0.05U DNase I，37℃下反应30min，随后热失活DNase I。
5)对DNase I酶切产物进行PCR扩增：将1.5μL步骤4)中得到的DNase I酶切产物溶液，20pmol正向引物，20pmol反向引物，1nmol dNTPs，1μL LC Green，0.5U Q5聚合酶充分混合于50μL Q5缓冲液中进行PCR扩增(程序为：98℃ 60s；98℃ 9s，63.5℃ 18s，72℃ 20s，循环35圈；72℃延伸600s)。
6)对步骤5)中PCR产物进行Sanger测序分析。
我们将人工合成的EGFR L858R野生型DNA和EGFR L858R突变型DNA的标准品按不同比例混合，分别得到初始突变丰度为0.1％和0.01％的样品。对两种突变丰度不同的样品分别采用步骤4)预处理不同时间(0min，18min，45min，90min)后再进行步骤5)PCR扩增和步骤6)Sanger测序，得到的Sanger测序图谱如6所示。不经硫代DNA引导DNase I酶切处理的样品(0min)，用Sanger测序无法检出目标位点突变峰的存在。经过处理的样品，在Sanger测序图谱中可检测到目标位点突变峰的信号。且随着硫代DNA引导DNase I酶切时间的增加，Sanger图谱中突变峰的信号逐渐增强，说明待测体系中突变链的丰度随着DNase I对野生链的选择性酶切而显著的提升，同时也证实了RNA封闭链对于底物链非目标区域的有效封闭。
在此基础上，我们将方法用于患者组织样品和血浆样品的分析。利用试剂盒提取纯化分别得到组织样品基因组DNA和血浆样品基因组DNA，采用步骤1)-步骤6)进行处理和检测，同时设置不进行步骤4)硫代DNA引导DNase I酶切处理的对照组和采用相同步骤进行处理的标准品的对照组，最终的Sanger测序图谱如图7所示。可见，未经步骤4)酶切处理的对照组仅组织样品基因组DNA的突变可以在Sanger测序图谱中看到，突变丰度约为30％。经过步骤4)硫代DNA引导DNase I酶切处理的实验组中，初始突变丰度为1％，0.1％和0.01％的样品在Sanger图谱中的突变峰信号分别达到约60％，30％和10％。血浆样品经处理后基因突变位点亦可被Sanger测序检出。肺癌阳性组织样品经过处理后在Sanger图谱中的突变丰度也明显升高。阴性组织样品经过处理后在Sanger图谱中未出现假阳性信号。
综合以上实验结果，本发明的方法使得Sanger测序法不能直接检测的低丰度基因突变样品在经过选择性酶切处理后可使突变链的丰度提升到直接由Sanger测序法测到，其中样品处 理过程只需4小时，从拿到临床样本到给出Sanger测序结果，整个分析过程时间不超过24小时，这为临床样品的快速测序分析提供了强有力的手段，具有良好的推广应用前景。

Claims (10)

  1. 一种基因突变检测方法，包括以下步骤：
    1)确定基因组DNA待测目标序列，该目标序列包括目标区域和非目标区域，设计并合成与野生型DNA序列目标区域互补的硫代DNA链以及与非目标区域互补的RNA封闭链；
    2)对待测目标序列进行PCR扩增，利用Lambda核酸外切酶将扩增产物处理成单链；
    3)将步骤2)得到的单链DNA与步骤1)合成的硫代DNA链和RNA封闭链在DNase I缓冲溶液中混合，升温退火后加入DNase I反应一段时间，热失活DNase I；
    4)对步骤3)酶切产物进行测序检测。
  2. 如权利要求1所述的基因突变检测方法，其特征在于，步骤1)中所述待测目标序列的长度为115～130nt。
  3. 如权利要求1所述的基因突变检测方法，其特征在于，步骤1)中所述待测目标序列上可能存在突变位点的目标区域位于待测目标序列的中部，目标区域的长度控制在其熔解温度Tm在42～46℃之间。
  4. 如权利要求3所述的基因突变检测方法，其特征在于，目标区域的长度为11～13nt。
  5. 如权利要求1所述的基因突变检测方法，其特征在于，步骤1)中所述非目标区域位于目标区域的两端，与非目标区域互补的RNA封闭链为多条，每条长度为25～90nt。
  6. 如权利要求1所述的基因突变检测方法，其特征在于，所述硫代DNA链的长度为16～59nt，包括与野生型DNA目标区域互补片段和两端的非互补片段。
  7. 如权利要求1所述的基因突变检测方法，其特征在于，所述硫代DNA链为全硫代DNA链。
  8. 如权利要求1所述的基因突变检测方法，其特征在于，步骤2)中PCR扩增采用的正向引物为5’-OH末端，反向引物的5’末端磷酸化标记，Lambda核酸外切酶选择性地消化双链DNA的5’磷酸化的链。
  9. 如权利要求1所述的基因突变检测方法，其特征在于，步骤2)中PCR扩增产物经Lambda核酸外切酶处理成单链后通过超滤纯化。
  10. 如权利要求1所述的基因突变检测方法，其特征在于，步骤3)将步骤2)得到的单链DNA与步骤1)合成的硫代DNA链和RNA封闭链在DNase I缓冲溶液中混合，升温退火后加入DNase I，在37～42℃温度下反应30～45min，然后热失活DNase I。
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