CN109680044B - Gene mutation detection method based on selective elimination of wild chain background interference - Google Patents

Abstract

The invention discloses a gene mutation detection method based on selective elimination of wild chain background interference. Amplifying a target sequence to be detected by PCR, and processing the target sequence into single-stranded DNA by Lambda exonuclease; designing and synthesizing a thio DNA chain complementary with a target region of a wild type DNA sequence and an RNA closed chain complementary with a non-target region, mixing with single-stranded DNA, heating and annealing, and adding DNase I for cutting; DNase I selectively excises a target region sequence of a wild type DNA chain under the guidance of a thioDNA chain, and a mutant type DNA chain cannot be excised by DNase I due to the mismatch in the target region and is reserved, so that the abundance of the mutant type DNA chain is remarkably improved, and the difficulty of detecting low-abundance gene mutation by the prior art is greatly reduced. The method of the invention does not need complex and expensive instruments, is easy to operate, has low cost, can give results within 1 day, and can provide timely and reliable gene mutation information for clinical tumor early screening and postoperative recurrence monitoring.

Description

Gene mutation detection method based on selective elimination of wild chain background interference

Technical Field

The invention relates to the field of genome sample treatment and gene mutation detection, in particular to a method for pretreating a genome sample.

Background

Cancer (malignancy) is today the first killer of serious threat to human health, and early diagnosis and monitoring of postoperative recurrence associated with cancer are of great biological and medical significance. Gene mutation is a heritable mutation phenomenon of genome DNA molecules, and is one of the important reasons for malignant tumor formation. The traditional tumor gene detection means is mainly tissue biopsy, namely a tumor tissue sample is obtained by a surgical puncture mode and is subjected to gene detection, the method is mainly used for detecting cancerous tissues, effective detection cannot be realized on early tumor cells which do not form focuses, and false negative detection results can be obtained on tumor tissues which form focuses due to tumor heterogeneity. In addition, such invasive detection means causes great pain to the patient and may further stimulate the cancerous tissue to deteriorate during surgical penetration. Tissue biopsy is also not suitable for continuous sampling and disease follow-up.

With the introduction of the concept of precision medicine and the circulation of tumor DNA (Circulating tumor DNA)¹The discovery of novel tumor markers such as circulating tumor cells and exosomes has led to the development of fluid biopsy as a new hope for early diagnosis of cancer. ctDNA is DNA released into the circulation after early tumor cell shedding or apoptosis, which is highly fragmented but carries all the genetic mutation information of tumor cells. The detection aiming at ctDNA in a circulating system can provide important information for early screening and relapse monitoring of tumors. Since normal cells also release a large amount of fragmented DNA into the circulatory system during apoptosis, a large degree of background interference is caused to the detection of ctDNA in plasma free DNA (cfDNA). The abundance of ctDNA with gene mutation in plasma is reported in the literature to be generally between 0.001% and 10%, which puts high demands on the sensitivity and selectivity of the mutation detection method.

At present, the detection method aiming at the gene mutation mainly comprises a sequencing method, a digital droplet PCR method, a DNA probe hybridization method and the like. The direct sequencing method is the most classical gene mutation detection method with the widest application range. The sequencing method can directly give the nucleotide sequence of the target DNA molecule, and is therefore considered as a gold standard for the detection of gene mutations. The Sanger sequencing method (dideoxy chain termination method) realizes low-cost and rapid detection of a single sample through decades of development and technical optimization, but has limited sensitivity, the minimum detection limit on the abundance of a mutant gene is between 5 and 10 percent, and the method cannot be directly used for the detection of ctDNA. Second generation sequencing (Next-Generation sequencing, NGS)²A principle distinct from the classical Sanger chain termination method is adopted, which implements Sequencing By Synthesis (SBS) in a massively parallel manner. Under the conventional sequencing depth, the mutation abundance range of the NGS capable of giving effective data is more than 0.1-0.5%. On this basis, Newman et al developed a tumor individualized depth sequencing analysis (Cancer characterized profiling by deep sequencing, CAPP-NGS). The method comprisesThe core idea is to narrow the detection range of NGS, screen out about 0.04% of gene library of target range through the analysis of large samples of human population, only carry out high-depth second-generation sequencing on the gene library, the detection limit can reach 0.02%, and simultaneously realize the simultaneous detection of hundreds of gene mutations. However, CAPP-NGS still can not meet the detection of early cancer samples, and in order to reach the detection limit of 0.02%, the sequencing depth of CAPP-NGS needs to be more than 10000 times, the detection cost is extremely high, the detection period reaches several weeks, and the wide-range popularization is difficult. While second generation sequencing was developed and commercialized, with nanopores³Sequencing is also gaining wide attention as a representative third generation sequencing technology. Nanopore sequencing utilizes α -hemolysin to construct a biological Nanopore that allows single-stranded DNA to pass right through the middle of the pore. When the DNA chain passes through the nanopore, other ions can be prevented from freely entering and exiting the nanopore, so that the current near the nanopore is changed, in addition, the chemical structures and the sizes of four basic groups on the DNA are slightly different, and the sequence information of the DNA can be indirectly obtained by monitoring the current near the nanopore in real time. The main features of the Nanopore technology are the surprising read length and small volume, up to 147Kb in practical applications. However, the accuracy of the sequencing result is not high enough at present, only 80%, and a certain distance is left from the direct sequencing of ctDNA.

Digital droplet PCR (digital multiplex PCR, ddPCR)⁴On the basis of the traditional PCR technology, the chip or the liquid drop is utilized to realize the independent distribution of the sample, the PCR is carried out on the independent liquid drop, the limit detection limit can reach 0.005-0.05 percent, and the method is the method with the highest sensitivity for detecting the ctDNA at present, but the requirement for the ultra-dispersion of the original PCR system to the micro-drop is extremely high when the limit detection limit is reached. In order to ensure the accuracy of the determination, the stable detection limit of the method is artificially controlled to be between 0.01 and 0.1 percent, and the detection limit is controlled to be between 0.05 and 0.1 percent during actual clinical use. The lower the limit of detection, the greater the amount of sample required. The key technical core of ddPCR is to realize the single-molecule-level dispersion of the DNA to be detected, the practical operation is complicated, the price of an instrument is high, the operation cost is high, the detection period is 2-4 days, and the rapid analysis of ctDNA is not easy to realize.

The fluorescent probe method using artificialThe designed and synthesized single-chain DNA probes respectively marked with a fluorescent group and a quenching group specifically identify and combine a mutation target chain to be detected in a system and give a fluorescent signal, thereby realizing the detection of the abundance of the mutation gene in a sample to be detected. Under general determination conditions, the difference between a wild strand and a mutant strand in a sample to be determined is only one base, the discrimination capability of a DNA probe for the base is limited, and the detection limit in the practical application process is about 10%. In order to further improve the sensitivity of the DNA fluorescent probe, a variety of novel probes such as molecular beacons, binary probes, three-stem probes, etc. have been developed based on simple probes, which reduces the detection limit to 3%. Zhang⁵Et al further introduced a large number of thermodynamic and kinetic calculations based on simple DNA hybridization probes, further reducing the detection limit to 0.01% to 1% using recognition of strand competition, strand displacement, strand migration, but this method is long in detection time and requires precise calculations for probe and competitor sequences. Das is⁶The gene mutation electrochemical detection method based on probe hybridization PCR is developed by combining a DNA probe and a high-sensitivity electrochemical detection method, and the method can directly measure 0.01% of mutation chains by means of the high sensitivity of electrochemical detection and abandons the traditional PCR, so that the detection level of the gene mutation with ultra-low abundance is achieved. However, the method has poor universality, the structure of the nano electrode and the synthesis of the nano carrier on the surface of the electrode are difficult, an electrochemical detection system needs to be redesigned according to different detection systems, and meanwhile, the cost of PNA used in a large amount in the system is high, so that the further popularization of the method is limited. Xiao⁷The sensitivity of the fluorescence probe method is obviously improved by introducing nuclease to assist the amplification of the fluorescence signal on the basis of the original fluorescence probe method. Fluorescence analysis lacks direct information on base changes compared to sequencing.

At present, the PCR amplification cannot be carried out for the detection of actual samples, and the method for preferentially enriching the mutant chains in a sample system by improving the selectivity of the PCR process is also widely concerned and researched. The replacement of the conventional PCR in the amplification stage with the selective PCR can improve the abundance of the mutant chain in the sample to be detected before the final measurement, thereby improving the abundance of the mutant chain in the sample to be detectedSensitivity of abundance mutations. The existing selective PCR method mainly includes mutation retardation Amplification PCR (Amplification feedback Amplification system PCR, ARMS PCR)⁸Wild-type blocking PCR (Wild-type blocking PCR)⁹Low temperature PCR (Co-amplification at low temperature PCR, COLD-PCR)¹⁰Locked nucleic acid/peptide nucleic acid Clamp PCR (Locked nucleic acids/peptide nucleic acids-mediated PCR, LNA/PNA-mediated PCR)¹¹And ARMS-qPCR and ice-COLD PCR which are combined by the above methods. However, the PCR system is a complex biochemical reaction involving multiple temperature combinations and multiple components, and is complicated in primer design, temperature optimization and time control, and the product composition is very complex. A new competitive component is additionally introduced in the PCR process, so that the reaction becomes more complicated and difficult to control. In the experimental process, the sequence of each nucleic acid chain, the adding amount of each component, the annealing temperature and the constant temperature time of each stage need to be subjected to complex optimization and precise control, and the influence factors are complex. Even in the case of the combinatorial selective PCR, the stable detection limit is controlled to be between 0.05% and 0.1%, and the requirement of rapid sequencing analysis of ctDNA is difficult to meet.

Because the wild type DNA has no mutation, does not carry relevant information of key pathogenic genes, can not provide key information for early screening of tumors or monitoring of postoperative recurrence, but occupies most proportion in a detection system, and brings huge background interference to the detection of mutant DNA in a sample. If a simple and efficient method capable of selectively removing wild-type DNA in a system can be constructed, the abundance of mutant-type DNA can be remarkably improved, and then the mutant-type DNA can be analyzed by a conventional sequencing method. In this respect, two groups have now conducted exploratory studies, each with the use of double-strand specific nucleases (DSN enzymes)¹²And magnetic bead capture (DISSECT)¹³The method of (1) selectively removes a large amount of wild type DNA from a sample to be tested. However, both methods have significant limitations and it is difficult to obtain stable and reliable results. For example, DSN enzymes themselves are not sequence selective for wild-type and mutant DNA, and their ability to discriminate between single-stranded and double-stranded substrates is derived solely from the substrate strand, aided by precise temperature control. This principle leads toLong-chain DNA of the genome may be non-specifically hydrolyzed, resulting in a failure of the enrichment. Another DISSECT technology is to remove wild type DNA in the system by using magnetic beads modified by complementary sequences through selective hybridization. Since the removal process is linear, the single-pass removal efficiency is low, and the non-specific adsorption on the surface of the magnetic beads easily results in the loss of the mutant DNA strands.

We have previously conducted studies to construct an enzyme complex that regulates nuclease sequence selectivity by electrostatic self-assembly of DNase I and thio DNA strands at high concentrations (patent No.: ZL 201410797398.6)¹⁴. The enzyme complex can efficiently and selectively excise a substrate strand which is completely complementary and paired with a thioDNA strand in a system, and lays an important foundation for developing a method for selectively removing a wild strand in a genome sample.

In conclusion, the existing method still has a series of problems of low sensitivity, long detection period, unstable method, strict requirements on experimental condition control, high price of instrument and reagent and the like in the aspect of early tumor detection aiming at ctDNA. Aiming at the problems, the invention aims to develop a gene mutation detection method which has high sensitivity, relatively simple operation, good result stability, lower cost, easy popularization and application and service for clinical examination.

Reference to the literature

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Disclosure of Invention

The invention aims to provide a gene mutation detection method, which removes wild type DNA in a system to be detected by constructing a simple and efficient method, so that the abundance of mutant strand DNA is increased to be directly detected by a Sanger sequencing method.

During the process of intensive mechanism research on the method established in the patent ZL 201410797398.6, the fact that the affinity between DNase I and a thio DNA chain is obviously greater than that between the DNase I and a natural DNA single chain is found. This means that in a mixed system DNase I will preferentially bind to thio DNA strands before interacting with normal DNA strands. By utilizing the characteristic, the invention constructs a method for preferentially combining the DNase I from the free thio DNA chain in the solution under the condition of low-concentration DNase I without pre-assembly and then inducing the DNase I to selectively cut the sequence which is completely complementary with the thio DNA chain in situ in the solution. According to the DNA sequence of a target gene to be detected, designing the sequence of a thioDNA chain to be completely complementary with wild type DNA, so that the thioDNA chain is rapidly hydrolyzed; the mutant strand is not substantially cleaved due to a single base mismatch with the thio DNA strand. The proportion of the mutant chain in the genome sample treated by the system is obviously improved, and the simplest Sanger sequencing method is directly used for detecting whether the mutation exists in the genome sample and the type of the mutant base. Further, by the quantitative relation between the processing time and the mutation enrichment multiple, the mutation abundance value in the original genome sample can be calculated.

The principle of the method of the present invention is shown in FIG. 1, and the region (10-12 bases) of the mutant DNA strand and the wild-type DNA strand where the mutation site may exist is designated as the target region, and the thioDNA strand sequence is artificially designed to be perfectly complementary to the target region of the wild-type DNA strand, while the target region of the mutant DNA strand cannot be perfectly complementary to the thioDNA strand sequence due to the mutation site. The sequences of the mutant DNA strand and the wild-type DNA strand outside the target region are designated as non-target regions, and the sequences of the mutant DNA strand and the wild-type DNA strand are identical in the non-target region. To prevent non-specific cleavage of the non-target region by DNase I, RNA single strands fully complementary to their sequences were added to the solution to block them.

When the above method is applied to analysis of a genomic sample (e.g., a plasma sample or a tissue sample), DNA therein is first extracted and purified using a kit, and then a target sequence (wild type or mutant type) to be detected in the extracted DNA is amplified by PCR. The amplification product is treated by Lambda exonuclease to form single-stranded DNA, which contains a large amount of wild type single-stranded DNA and a small amount of mutant type single-stranded DNA. Adding an RNA closed chain and a thio DNA chain into the system, heating and annealing, and then adding DNase I for cutting. DNase I is guided by thio-DNA to selectively excise the target region sequence of the wild-type DNA strand, and the mutant DNA strand cannot be excised by DNase I due to the mismatch in the target region and is retained in a solution system. Along with the enzyme digestion reaction, the abundance of the mutant DNA chain is remarkably improved until the range which can be accurately detected by the conventional mutation detection method, and the mutant DNA chain can be directly detected by PCR amplification and Sanger sequencing.

The gene mutation detection method of the invention specifically comprises the following operation steps:

1) determining a target sequence to be detected of genome DNA, wherein the target sequence comprises a target region and a non-target region, and designing and synthesizing a thioDNA chain complementary with the target region of a wild type DNA sequence and an RNA closed chain complementary with the non-target region;

2) designing a PCR amplification system to amplify a target sequence (including a wild type and a mutant type) to be detected, and processing an amplification product into a single strand by using Lambda exonuclease;

3) mixing the single-stranded DNA obtained in the step 2) with the thio-DNA chain synthesized in the step 1) and the RNA closed chain in a DNase I buffer solution, heating and annealing, adding DNase I to react for a period of time, and thermally inactivating the DNase I;

4) sequencing and detecting the enzyme digestion product in the step 3).

In the step 1), the length of the target sequence to be detected is preferably 115-130 nt, and a target region in which a mutation site may exist is preferably located in the middle of the target sequence to be detected. One or more mutation sites may exist in the target region, the length of the target region is controlled to be within a melting temperature Tm of 42-46 ℃ according to the GC content of the sequence, and generally, the length of the target region is preferably 11-13 nt.

The non-target regions are positioned at two ends of the target region, the length of the RNA closed chain which is complementary with the non-target regions is usually 25-90 nt, and if the non-target regions are longer, a plurality of RNA closed chains can be designed and synthesized.

The length of the thio DNA chain is preferably 16-59 nt, and the thio DNA chain comprises a fragment complementary to a wild type DNA target region and non-complementary fragments at two ends. Preferably, the thio DNA strand is a fully thio DNA strand.

In the step 2), preferably, the forward primer of the PCR amplification system is a 5 ' -OH end, the 5 ' end of the reverse primer is phosphorylated, and the Lambda exonuclease selectively digests the 5 ' phosphorylated strand of the double-stranded DNA. The amplification product is treated into single strand by Lambda exonuclease and then purified by ultrafiltration.

After PCR amplification in the step 2), firstly carrying out ultrafiltration desalting on the PCR solution, then adding Lambda exonuclease, carrying out constant-temperature reaction at 37 ℃ for a certain time, then thermally inactivating the Lambda exonuclease, and then carrying out ultrafiltration purification on the enzyme digestion product solution again.

And 3) mixing the single-stranded DNA obtained in the step 2) with the thio-DNA chain and the RNA closed chain synthesized in the step 1) in a DNase I buffer solution, heating and annealing, adding DNase I, reacting at the temperature of 37-42 ℃ for 30-45 min, and then thermally inactivating the DNase I. Wherein the condition of temperature-rising annealing is that the materials are melted at 95 ℃ for more than 30s and then slowly cooled to room temperature, and the cooling rate is not more than 10 ℃/min, so that incomplete hybridization is prevented. The specific procedure may be: 90s at 95 ℃; 90s at 80 ℃; 90s at 65 ℃; 90s at 50 ℃; 120s at 37 ℃. The method for detecting the DNase I enzyme digestion product in the step 4) comprises but is not limited to the following steps: sanger sequencing detection, pyrosequencing, next-generation sequencing and the like after PCR amplification.

The thio-DNA strand and the RNA-blocked strand used in the invention are both obtained by chemical synthesis, wherein the thio-DNA strand is not hydrolyzed by nuclease, the RNA-blocked strand is not cut by DNase I, and the DNA in the hybrid strand can be inhibited from being cut by DNase I after hybridizing with the DNA single strand in the non-target region.

Compared with the prior art, the invention has obvious technical advantages, which are specifically set forth as follows:

two types of mainstream genetic mutation detection technologies currently in use are second-generation sequencing and digital droplet PCR. The second generation sequencing method has the problems that the detection period is long, especially for low-abundance gene mutation samples, longer sequencing and data processing are needed, usually 2-4 weeks, the cost is high, and the requirement of rapid detection of a large amount of clinical samples is difficult to meet. The digital droplet PCR method relies on a large sample size to provide a sufficiently high sensitivity, which is unacceptable to the patient. According to the invention, wild type DNA is removed through one-step pretreatment, so that the mutation abundance is increased to a range which can be detected by a Sanger sequencing method. Experiments prove that the method can still obtain obvious enrichment effect on samples with mutation abundance as low as 0.01 percent, thereby greatly reducing the difficulty of detecting low-abundance gene mutation in the prior art. The whole method does not need complex and expensive instruments, is easy to operate and low in cost, can give results within 1 day, has a practical application value compared with a second-generation sequencing method, and can provide timely and reliable gene mutation information for clinical tumor early screening and postoperative recurrence monitoring.

Drawings

FIG. 1 is a schematic diagram of a method for direct Sanger sequencing after enrichment of low-abundance gene mutation samples by cutting wild type DNA with DNase I guided by a thio DNA chain to remarkably improve abundance of mutant type DNA.

FIG. 2 is a graph showing a comparison of the relative rates of digestion by DNase I after hybridization of the wild-type probe and the mutant-type probe of EGFR L858R with different kinds of nucleic acid strands in example 1.

FIG. 3 is a fluorescence plot of DNase I cleavage reaction of EGFR L858R wild-type probe (a) and mutant probe (b) under the guidance of different concentrations of thio DNA strands in example 1, wherein the inset in (b) is an enlarged view of four fluorescence rising curves.

FIG. 4 is a gel electrophoresis of the products of EGFR L858R wild-type long-chain DNA and mutant long-chain DNA cleaved with thioDNA chain-directed DNase I for 30min after RNA chain blocking in example 1 at different temperatures.

FIG. 5 is the gel electrophoresis of the product of KRAS G13D wild type long-chain DNA and mutant long-chain DNA cleaved for 30min after RNA chain blocking by DNase I guided by three thio DNA chains of different lengths in example 1.

FIG. 6 is a Sanger sequencing spectrum of PCR products of samples with 0.1% and 0.01% abundance of EGFR L858R mutation in example 2 after various times of thioDNA-guided DNase I pretreatment.

FIG. 7 is a Sanger sequencing spectrum of PCR products before and after thioDNA-guided DNase I pretreatment of standard and negative tissue samples, positive tissue samples of lung cancer patients and plasma samples with EGFR L858R mutation abundances of 1.0%, 0.1% and 0.01% in example 2.

Detailed Description

The invention will be further elucidated by means of specific embodiments in the following description, in conjunction with the appended drawings. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 Selective excision of wild type DNA Strand by thioDNA Strand guided DNase I >

In this example, two different target gene DNA sequences were selected, one being the sequence of point mutation at codon 858 of exon 21 of human Epidermal Growth Factor Receptor (EGFR) (L858R); the other is the sequence of the point mutation (G13D) at codon 13 of the mouse sarcoma virus oncogene of V-Ki-ras2Kirsten (KRAS) No. 2 exon.

TABLE 1 sequence of nucleic acid strand used in this example (5 '-3')

(1) EGFR L858R thio DNA chain leading DNase I selectively cutting EGFR L858R wild type DNA chain

EGFR L858R wild-type long-chain DNA andthe sequence of EGFRL858R mutant long-chain DNA is shown in Table 1, the underlined part is the target region, and the mutation site is shown in bold. Aiming at the target region, designing a corresponding EGFR L858R thio DNA chain sequence as follows: 5' -AAAAAAAAAAAGGGCTGGCCAACGCAGATA-3', wherein the phosphate backbone is fully thio-modified, the 11-nt sequence fully complementary to the wild-type target sequence is underlined, and the rest is for increasing the binding force with DNase I.

In order to show the difference of the cleavage rate of the thioDNA sequence to the wild strand and the mutant strand more intuitively, we synthesized the fluorescent labeled wild type and mutant short target sequences with the length of 16nt, which are respectively named as EGFR L858R wild type probe and EGFR L858R mutant probe (see the sequence in Table 1), wherein the EGFR L858R wild type probe has a complete complementary pairing with the EGFR L858R thioDNA strand with 11nt sequence, and the EGFR L858R mutant probe has a one-to-one base mismatch with the EGFR L858R thioDNA strand with the corresponding 11nt sequence.

The two probes are both marked with a fluorescent group and a quenching group, and high-efficiency fluorescence resonance energy transfer can occur between the two probes. When the two probe sequences are kept intact, the fluorescence emitted by the excited fluorescent group is absorbed by the quenching group, so that no fluorescence can be detected. When the sequences are cut by DNase I, the quenching groups are far away from the fluorescent groups, and the solution emits strong fluorescence, so that whether the probe sequences are selectively cut by the DNase I guided by the thio DNA and the speed and the progression degree of the cutting reaction can be effectively indicated.

The specific implementation steps are as follows:

1) 20pmol of wild-type probe or mutant-type probe and 20pmol of the thioDNA strand were mixed in 50. mu.L of a buffer solution (composition: 10mmol/L Tris-HCl,2.5mmol/L MgCl)₂,0.5mmol/L CaCl₂pH 7.6@25 deg.C), and then heating and annealing the solution (the specific procedure is 95 deg.C 90 s; 90s at 80 ℃; 90s at 65 ℃; 90s at 50 ℃; 37 ℃ for 120 s).

2) Adding 0.05U DNase I into the solution annealed in the step 1), uniformly mixing, immediately putting into a real-time fluorescence PCR instrument (Rotor Gene Q), and measuring the fluorescence value at 37 ℃, wherein the excitation wavelength is 470 +/-10 nm, and the detection wavelength is 510 +/-5 nm.

A total of 4 control experiments were performed using wild type and mutant probes according to the above procedure, wherein the first group did not contain a thio DNA strand; the second group replaced the thiodna strand with the EGFR L858R RNA complementary strand that was perfectly complementary paired with the wild-type probe (see table 1 for sequence); the third group replaced the thio DNA strand with the complementary strand of EGFR L858R DNA that was perfectly complementary paired with the wild-type probe (see Table 1 for sequence); the fourth group was prepared by adding the previously described EGFR L858R thio DNA strand perfectly complementary to the wild-type probe in accordance with the normal procedure. The rates of fluorescence increase by enzymatic hydrolysis of the wild-type probe and the mutant-type probe obtained in the four experiments are shown in FIG. 2.

It can be seen that both probes are themselves cleaved by DNase I at a slow rate and there is no significant difference between the two. After the RNA complementary strand is added, a DNA/RNA hybrid strand can be formed with both probes, and the rates of the two probes for being cut by DNase I are slow and have small difference. After the third group of experiments add DNA complementary strands, the wild type probe forms a completely complementary paired DNA double strand, the mutant type probe forms a single-base mismatched DNA double strand, both DNA double strands are rapidly hydrolyzed by DNase I, and the cutting rate of the double strand formed by the mutant type probe is even slightly faster than the completely complementary double strand formed by the wild type probe; after the thio DNA chain is added in the fourth group of experiments, the wild type probe and the wild type probe are completely complementary and paired to form a DNA-thio DNA hybrid chain, so that the wild type probe and the wild type probe are rapidly cut by DNase I, and the mutant type probe is cut by the DNase I at a rate even slightly less than that of the mutant type probe due to the single-base mismatch with the thio DNA chain. The control results of these four experiments fully demonstrated that thio-DNA can direct DNase I to selectively cleave the wild-type DNA strand that is perfectly complementary paired to the thio-DNA sequence, while having substantially no effect on the mutant DNA strand where only a single base mismatch is present.

To further confirm the priming effect of thio-DNA, we further designed four control experiments. The addition amounts of the wild-type probe and the mutant-type probe in each set of experiments were 20pmol, the addition amounts of the thioDNA strands from the first set to the fourth set were 0, 1pmol, 10pmol and 25pmol in this order, the final concentrations of the corresponding thioDNA strands were 0, 20nM, 200nM and 500nM, respectively, and the results of real-time fluorescence analysis are shown in FIG. 3.

As can be seen from the data in FIG. 3(a), the rate of cleavage by DNase I of the wild-type probe gradually increased with increasing amounts of thioDNA strands added. When the amount of the added thio DNA strand is larger than that of the wild-type probe, the fluorescence signal quickly reaches a platform, which indicates that the wild-type probe is quickly removed by DNase I hydrolysis. The mutant probe shows the opposite result, as shown in fig. 3(b), as the addition amount of the thio-DNA strand increases, the rate of the mutant probe being cleaved by DNase I becomes slower and slower, further, the binding force between the thio-DNA strand and DNase I is greater than that between the ordinary DNA single strand and DNase I, and after the thio-DNA strand and DNase I are preferentially bound, the mutant probe is not easily bound directly to DNase I, nor easily cleaved by hybridization with the thio-DNA strand, and thus is retained instead.

The wild-type probe and the mutant-type probe used in the fluorescence analysis experiment are both 16nt short-chain DNAs, and in an actual genome sample system, the DNA to be detected is usually long-chain. To prevent non-specific cleavage by DNase I due to the formation of secondary structures by itself or partial hybridization between strands in the target region of the long DNA strand, we protected it by adding the RNA complementary strand of the non-target region to the solution, as shown in FIG. 2 for the second set of experiments. The sequences of the wild-type and mutant long-chain DNAs of EGFR L858R are shown in Table 1, and the sequence length is 130nt, wherein the target region complementary to the thio DNA is 11nt, and the rest is non-target region. Based on the length limitation of RNA that can be synthesized in large quantities at present, we designed 4 RNA-blocked strands (see Table 1 for sequence) with length between 25nt and 30nt for blocking the non-target region part of long-chain DNA sequence. Because the long-chain DNA sequence is not marked with a fluorescent group, the condition of a product of DNase I cutting the long-chain DNA under the guiding of a thioDNA chain and the closing of an RNA chain is represented by a gel electrophoresis method. The specific experimental steps are as follows:

1) EGFR L858R wild-type long-chain DNA or mutant long-chain DNA (20pmol), EGFR L858R RNA-blocked strands 1-4 (20pmol each), EGFR L858R thio DNA strand (25pmol) were mixed in 50. mu.L of buffer solution consisting of: 10mmol/L Tris-HCl,2.5mmol/L MgCl₂,0.5mmol/L CaCl₂And heating and annealing at the pH of 7.6@25 ℃.

2) Control experiments in which no DNase I was added, 0.05U of DNase I was added, and the reaction was carried out at different temperatures (33.3 ℃ to 45.9 ℃) after the addition of the enzyme were designed for both the wild-type long-chain DNA and the mutant long-chain DNA, and the reaction time was 30min, followed by heating to inactivate the DNase I. A small amount of the product solution (9. mu.L) was subjected to agarose gel electrophoresis.

3) Ten sets of 2.5% agarose gel electrophoresis experiments were designed, in which lane 1: adding an RNA closed chain and a thio DNA chain into a wild type DNA long chain, and adding no DNase I after heating and annealing; lanes 2-5: adding a wild type DNA long chain into an RNA closed chain and a thio DNA chain, heating and annealing, and then treating for 30min by DNase I, wherein the reaction temperature is 33.3 ℃, 37.1 ℃, 41.7 ℃ and 45.9 ℃ in sequence; lane 6: adding RNA closed chain and thio DNA chain into the mutant DNA long chain, and carrying out heating annealing without adding DNase I treatment; lanes 7-10: adding RNA closed chain and thio DNA chain into the mutant DNA long chain, heating and annealing, and treating with DNase I for 30min at 33.3 deg.C, 37.1 deg.C, 41.7 deg.C and 45.9 deg.C. DNA band distribution was observed after staining with GelRed nucleic acid dye.

The results of the above gel electrophoresis are shown in FIG. 4. Comparing lane 1 and lane 6, it can be seen that in the absence of DNase I, both the long wild-type DNA strand and the long mutant DNA strand bind to 4 RNA-blocked strands to form a hybrid duplex of 130bp in length. Comparing lanes 1-5, it can be seen that, at four different reaction temperatures, the substrate strand for hybridization formed by the long wild-type DNA strand and the RNA closed strand can be rapidly digested by DNase I, and the digestion products have lengths of about 80bp and 30bp, which correspond to the double strands of hybridization formed by the target region outside the target regions at the 5 'end and the 3' end of the substrate strand and the RNA closed strand, respectively. The results show that hybridization of the RNA-blocked strand effectively prevents DNase I from cleaving non-target regions, and does not affect the efficient cleavage of target regions by DNase I guided by the thio-DNA strands in the system. Comparing lanes 6-10, it can be seen that the hybridization substrate formed by the long mutant DNA strand and the RNA blocking strand was hydrolyzed by DNase I at 33.3 ℃ for 30 min. As the temperature increases, the stability of the hybridization product decreases and it is increasingly hydrolyzed by DNase I. When the temperature is raised to 45.9 ℃, most of the hybridization product is hydrolyzed. Compared with the product bands of the long chain of wild-type DNA shown in lanes 1-5, the hydrolysis products of the long chain of mutant DNA are not concentrated in two distinct bands, indicating that the hydrolysis process is not selective and occurs randomly. The above experimental results show that after the non-target region of the mutant long-chain DNA is blocked and protected by the RNA strand, the target region can still be selectively cut by DNase I guided by the thio-DNA strand.

(2) KRAS G13D thio DNA chain guide DNase I selectively cut KRAS G13D wild type DNA chain

In order to prove the universality of the property that a thioDNA chain can guide DNase I to selectively excise a long target region sequence of wild type DNA, the sequence of a KRAS G13D gene mutation site is further selected for research. The sequences of KRAS G13D wild-type long-chain DNA, KRAS G13D mutant long-chain DNA, and KRAS G13D RNA blocked strands 1-4 for both strand non-target regions are shown in table 1. In addition, we designed and synthesized three different lengths of thio DNA strands for comparison, KRAS G13D thio DNA strands A (41nt), B (42nt) and C (43nt), respectively, with complementary pairing sequence lengths of 10nt, 11nt and 12nt, respectively, with the target region of the wild-type DNA strand.

The agarose gel electrophoresis experiment is used for comparing the effect of selectively cutting a target region sequence in a long-chain substrate by three different thio DNA chains to guide DNase I, and the specific operation steps are as follows:

1) KRAS G13D wild-type long-chain DNA or KRAS G13D mutant long-chain DNA (20pmol), KRAS G13D RNA-blocked strands 1-4 (20pmol each), KRAS G13D thio-DNA strand A/B/C (25pmol) were mixed in 50. mu.L of a buffer consisting of: 10mmol/L Tris-HCl,2.5mmol/L MgCl₂,0.5mmol/L CaCl₂And heating and annealing at the pH of 7.6@25 ℃.

2) According to the experimental design, 0.05U of DNase I is added into the experimental group, DNase I is not added into the control group, the enzyme digestion is carried out for 30min at 37 ℃, and the DNase I is thermally inactivated after the enzyme digestion is finished.

3) The DNA band distribution of the product solution in 2) was determined by 2.5% agarose gel electrophoresis.

Eight groups of 2.5% agarose gel electrophoresis experiments were designed and observed after staining with GelRed nucleic acid dye. Lanes 1, 3, 5, and 7 correspond to the detection results of the wild-type long-chain DNA, and lanes 2, 4, 6, and 8 correspond to the detection results of the mutant-type long-chain DNA. Lanes 1-2: DNase I is not added; lanes 3-4: adding a thio DNA chain A and DNase I for treatment for 30 min; lanes 5-6: adding a thio DNA chain B and DNase I for treatment for 30 min; lanes 7-8: add thioDNA strand C and DNase I for 30 min. The results of the experiment are shown in FIG. 5. As can be seen from comparison of lanes 1-8, after the long wild-type DNA chain and the long mutant-type DNA chain are blocked by the RNA blocking strand, the three thio-DNA chains can guide DNase I to selectively cut the target region sequence of the long wild-type DNA chain.

The research results show that in the practical application process, the sequence of the thioDNA chain can be flexibly designed according to the target region sequence of the substrate chain to be processed.

Example 2 Rapid sequencing analysis of Low abundance EGFR L858R Gene mutations by excision of wild-type DNA strands >

The sequences of the long wild-type DNA strand, the long mutant DNA strand, the thio-DNA strand and the RNA-blocked strand of EGFR L858R used in this example are shown in Table 1. The sequences of the PCR amplification primers used were:

EGFR L858R forward primer: 5'-TTCTTTCTCTTCCGCACC-3' (5 ' -OH end) (SEQ ID No: 21)

EGFR L858R phosphorylated reverse primer: 5' -PO₄-TACTTGGAGGACCGTCG-3 '(5' end phosphorylation marker) (SEQ ID No: 22)

The experimental procedure was as follows:

1) the genomic DNA was subjected to enzyme digestion with Sherase enzyme: 1mg of genomic DNA and 1.5. mu.L of Shearase enzyme were thoroughly mixed in 20. mu.L of a buffer consisting of 10mmol/L Tris-HCl, 25mmol/L MgCl₂1mmol/L DTT, pH 7.5@25 ℃ for 15min at 42 ℃ followed by heat inactivation of the Sherase enzyme.

2) Performing PCR amplification on the genome DNA and the standard product DNA: 1.5. mu.L of the product solution from step 1) or 0.25amol of DNA standard, 20pmol of forward primer, 20pmol of reverse primer, 1nmol of dNTPs, 1. mu.L of LC Green, 0.5U Q5 polymerase were mixed well in 50. mu. L Q5 buffer for PCR amplification (procedure: 60s at 98 ℃; 9s at 98 ℃, 18s at 63.5 ℃ and 20s at 72 ℃ for 35 cycles; extension at 72 ℃ for 600 s).

3) Single-stranded and purified PCR amplification product: subjecting the solution in step 2) to ultrafiltration desalination (ultrafiltration solvent: deionized enzyme-free water, the cutoff molecular weight of an ultrafiltration tube is 30KDa, the ultrafiltration temperature is 4 ℃, and the ultrafiltration time is as follows: 10min, rotating speed: 6000rpm, number of ultrafiltration: 2 times). The upper layer solution in the ultrafiltration tube was taken out and placed in a PCR tube (total volume of liquid was about 50. mu.L), 5U of Lambda exonuclease was added thereto, and the reaction was carried out at 37 ℃ for 10 minutes, followed by heat inactivation of Lambda exonuclease. And then, carrying out ultrafiltration purification on the enzyme digestion product solution again, wherein the ultrafiltration method is the same as the above method, and the content of the DNA in the upper layer solution after ultrafiltration is determined by using the Qubit 3.0.

4) The wild-type DNA strand was selectively excised using thio DNA strand-directed DNase I: mixing the genomic DNA or the standard DNA (1pmol) obtained in step 3), the RNA-blocked strand (10 pmol each) and the thioDNA strand (12.5pmol) in 50. mu.L of a buffer solution consisting of: 10mmol/L Tris-HCl,2.5mmol/L MgCl₂,0.5mmol/L CaCl₂pH 7.6@25 ℃, adding 0.05U of DNase I into the solution after temperature-rising annealing, reacting at 37 ℃ for 30min, and then thermally inactivating DNase I.

5) Performing PCR amplification on the DNase I enzyme digestion product: mu.L of the DNase I cleavage product solution obtained in step 4), 20pmol of forward primer, 20pmol of reverse primer, 1nmol of dNTPs, 1. mu.L of LC Green, 0.5U Q5 polymerase were mixed well in 50. mu. L Q5 buffer for PCR amplification (procedure: 60s at 98 ℃; 9s at 98 ℃, 18s at 63.5 ℃ and 20s at 72 ℃ for 35 cycles; extension at 72 ℃ for 600 s).

6) Sanger sequencing analysis was performed on the PCR products in step 5).

We mixed the artificially synthesized EGFR L858R wild-type DNA and EGFR L858R mutant-type DNA standards in different proportions to obtain samples with initial mutation abundances of 0.1% and 0.01%, respectively. After the two samples with different mutation abundances are respectively pretreated for different time (0min, 18min, 45min and 90min) in the step 4), the PCR amplification in the step 5) and the Sanger sequencing in the step 6) are carried out, and the obtained Sanger sequencing spectrum is shown as 6. Samples treated without thioDNA-directed DNase I digestion (0min) were not detectable for the presence of the target site mutation peak by Sanger sequencing. The processed sample can detect the signal of the target site mutation peak in a Sanger sequencing map. And with the increase of the enzyme digestion time of DNase I guided by the thioDNA, the signal of a mutation peak in a Sanger map is gradually enhanced, which shows that the abundance of a mutation chain in a system to be tested is obviously improved along with the selective enzyme digestion of the DNase I on a wild chain, and simultaneously, the effective closure of an RNA closed chain on a non-target region of a substrate chain is also verified.

On this basis, we used the method for the analysis of patient tissue samples and plasma samples. Extracting and purifying by using a kit to respectively obtain tissue sample genome DNA and plasma sample genome DNA, processing and detecting by adopting steps 1) -6), and simultaneously setting a control group which does not carry out step 4) thioDNA guide DNase I enzyme digestion treatment and a control group of a standard substance which carries out treatment by adopting the same steps, wherein the final Sanger sequencing map is shown in figure 7. As can be seen, the mutation of only the genomic DNA of the tissue sample in the control group which was not digested in step 4) can be seen in the Sanger sequencing map, and the abundance of the mutation is about 30%. In the experimental group treated by the step 4) thioDNA-guided DNase I enzyme digestion, the initial mutation abundance is 1%, and the mutation peak signals of 0.1% and 0.01% of samples in the Sanger map respectively reach about 60%, 30% and 10%. The gene mutation sites of the plasma samples after treatment can also be detected by Sanger sequencing. The mutation abundance of the lung cancer positive tissue sample in the Sanger map is obviously increased after the lung cancer positive tissue sample is treated. Negative tissue samples were processed without false positive signals in the Sanger profile.

By integrating the experimental results, the method provided by the invention can improve the abundance of the mutant chain to be directly detected by the Sanger sequencing method after the low-abundance gene mutation sample which cannot be directly detected by the Sanger sequencing method is subjected to selective enzyme digestion treatment, wherein the sample treatment process only needs 4 hours, and the whole analysis process time is not more than 24 hours from the time of taking a clinical sample to the time of giving the Sanger sequencing result, so that a powerful means is provided for the rapid sequencing analysis of the clinical sample, and the method has a good popularization and application prospect.

SEQUENCE LISTING

<110> Beijing university

<120> gene mutation detection method based on selective elimination of wild chain background interference

<130> WX2019-03-022

<160> 22

<170> PatentIn version 3.5

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Claims (9)

1. A method for detecting gene mutation for non-disease diagnostic purposes, comprising the steps of:

1) determining a target sequence to be detected of genome DNA, wherein the target sequence comprises a target region and a non-target region, designing and synthesizing a thioDNA chain complementary with the target region of a wild type DNA sequence and an RNA closed chain complementary with the non-target region, wherein the target region possibly provided with a mutation site on the target sequence to be detected is positioned in the middle of the target sequence to be detected, and the length of the target region is controlled to be 42-46 ℃ at the melting temperature Tm;

2) carrying out PCR amplification on a target sequence to be detected, and processing an amplification product into a single strand by using Lambda exonuclease;

3) mixing the single-stranded DNA obtained in the step 2) with the thio-DNA chain synthesized in the step 1) and the RNA closed chain in a DNase I buffer solution, heating and annealing, adding 0.05U of DNase I into a 50 mu L system for reaction for a period of time, and thermally inactivating the DNase I;

4) sequencing and detecting the enzyme digestion product in the step 3).

2. The method for detecting gene mutation according to claim 1, wherein the length of the target sequence to be detected in step 1) is 115 to 130 nt.

3. The method of detecting gene mutation according to claim 1 wherein the length of the target region is 11 to 13 nt.

4. The method for detecting gene mutation according to claim 1, wherein the non-target region in step 1) is located at both ends of the target region, and the number of RNA closed strands complementary to the non-target region is plural, each of which has a length of 25 to 90 nt.

5. The method for detecting gene mutation according to claim 1 wherein the thio-DNA strand has a length of 16 to 59nt and comprises a complementary fragment to the target region of the wild-type DNA and non-complementary fragments at both ends.

6. The method for detecting gene mutation according to claim 1 wherein said thio DNA strand is a fully thio DNA strand.

7. The method for detecting gene mutation according to claim 1, wherein the forward primer used for PCR amplification in step 2) is a 5 ' -OH terminal, the 5 ' -terminal of the reverse primer is a phosphorylated tag, and Lambda exonuclease selectively digests the 5 ' -phosphorylated strand of the double-stranded DNA.

8. The method for detecting gene mutation according to claim 1 wherein the PCR amplification product in step 2) is purified by ultrafiltration after being treated to single strand by Lambda exonuclease.

9. The method for detecting gene mutation according to claim 1, wherein the single-stranded DNA obtained in step 3) is mixed with the thio-DNA strand synthesized in step 1) and the RNA-blocked strand in a DNase I buffer solution, DNase I is added after annealing at a raised temperature, the mixture is reacted at 37 to 42 ℃ for 30 to 45min, and then DNase I is thermally inactivated.

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