Method for targeted enrichment of polymorphic sites by introducing thio-modified nucleic acid into single base

Abstract

The invention discloses a method for targeted enrichment of polymorphic sites by introducing thio-modified nucleic acid into a single base. According to the invention, the selector contains a segment of primer extension binding region with more than four basic groups, when the selector is extended by taking a target DNA / RNA as a template, single thio-modified dNTP and another three terminated dNTP / ddNTP, denaturation and renaturation are carried out, a hairpin structure is formed by itself, the selector without thio-modification is digested under the action of T4DNA polymerase, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA. And complementing the sequence at the incision to form an intermediate product with a hairpin structure, so as to fulfill the aim of enriching rare mutation sites. The method is suitable for DNA and RNA templates, and the obtained hairpin ring structure intermediate can be subjected to signal amplification in two ways: enzyme-mediated nucleic acid amplification or PCR amplification after linearization of a ring structure.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and in particular relates to a method for introducing a single base into a thio-modified nucleic acid for targeted enrichment of polymorphic sites. Background Art

[0002] Tumor histological specimens are the most commonly used sample type in clinical practice and are considered the gold standard for tumor molecular testing. However, the availability of sufficient histological specimens for molecular testing remains a significant obstacle to the widespread implementation of molecular pathology testing for lung cancer. Therefore, in addition to traditional tumor histological specimens, studies have also explored the use of various cytological supernatant specimens for molecular testing, such as body cavity fluids, sputum, bronchoalveolar lavage fluid (BALF), and fine needle aspiration specimens.

[0003] Nucleic acids circulating in body fluids but free from cells are called free nucleic acids, including cfDNA and cfRNA. cfDNA is the most widely detected target in liquid biopsies, especially circulating tumor DNA (ctDNA) that is free in body fluids. ctDNA in peripheral blood contains information on genetic variations, such as mutations in oncogenes and tumor suppressor genes, microsatellite instability, and epigenetic mutations. Currently, this information is primarily detected and analyzed through high-throughput sequencing technology and applied clinically.

[0004] As research continues to deepen, researchers have discovered that simple ctDNA testing cannot fully reflect the biological activity of the disease at the RNA level, nor can it clearly define the impact of mutations on cellular processes. However, RNA-level testing can pinpoint changes occurring at specific times in cancer, allowing for regular monitoring of disease progression and response to treatment, and predicting how different individuals with the same cancer will respond to different therapies. Consequently, this approach has garnered widespread attention.

[0005] For cfRNA, since the reverse transcriptases currently used cannot perform high-temperature cyclic amplification, the detection of cfRNA is not the optimal solution. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a method for introducing a single base into a thiophosphate-modified nucleic acid for targeted enrichment of polymorphic sites. This method involves only single-base extension, and the primer is detached from the template by increasing the temperature (below the inactivation temperature of the reverse transcriptase).

[0007] The present invention is achieved by introducing a single base into a thiothioate-modified nucleic acid for targeted enrichment of polymorphic sites, the method comprising the following steps:

[0008] (1) selecting a selector with a characteristic structure as a primer, and performing multiple rounds of single primer extension using the nucleic acid sample to be tested as a template under the conditions of a DNA synthesizer and its corresponding buffer and a single thio-modified base;

[0009] (2) The extension product obtained in step (1) was purified using a DNA purification kit, and then heated for denaturation at 90° C. for 1 minute, and placed on ice for 2 minutes for intramolecular renaturation to form an intermediate nucleic acid molecule with a hairpin structure having a single-stranded region at the 5′ end;

[0010] (3) T4 DNA polymerase is added to remove the selector without the introduction of the thiolated modified base, and the gap sequence is filled in by the action of T4 DNA polymerase to form a hairpin-shaped intermediate product;

[0011] (4) using the intermediate product as a template for nucleic acid amplification and subsequent chip or next-generation sequencing analysis.

[0012] Preferably, in step (1), a selector with a characteristic structure is selected as a primer, and multiple rounds of single primer extension are performed using the nucleic acid sample to be tested as a template under the conditions of DNA synthesizer and its corresponding buffer, a single thio-modified base, and a mixture of three other terminating dNTPs.

[0013] Preferably, the single thio-modified base is selected from one of dATPɑS, dGTPɑS, dCTPɑS, and dTTPɑS.

[0014] Preferably, if the method is used to enrich allele information, in step (1), the selector of the characteristic structure consists of four parts, which are: common primer 1 region, which consists of 18 to 30 bases; the mutant base complementary base extended by the specific primer; the primer extension binding region, which consists of 4 to 6 bases, and its sequence is in a reverse complementary relationship with the last base at the 3' end of the specific primer; common primer 2 region, which consists of 18 to 30 bases; the specific primer region for targeted enrichment of SNP site information, which consists of 18 to 30 bases, and the first base extended is the SNP site base.

[0015] Preferably, if the method is used to enrich allele information, in step (1), the selector of the characteristic structure consists of four parts, which are: a common primer 1 region consisting of 18 to 25 bases; a base complementary to the mutant base extended by the specific primer; a primer extension binding region consisting of 4 bases, the sequence of which is in a reverse complementary relationship with the last base at the 3' end of the specific primer; a common primer 2 region consisting of 18 to 25 bases; a specific primer region for targeted enrichment of SNP site information, consisting of 18 to 25 bases, and the first base extended is the SNP site base.

[0016] Preferably, when the method is used to enrich mutant gene information, in step (1), the sequences of common primer region 1 and common primer region 2 are the same in all selectors, which are primer sequences for PCR amplification of mutation site information and are used for high-throughput sequencing template preparation.

[0017] Preferably, in step (1), the nucleic acid sample is DNA or RNA, wherein, depending on the nucleic acid sample, the DNA synthesizing enzyme is TaqDNA polymerase that uses DNA as a template or reverse transcriptase that synthesizes DNA using RNA as a template.

[0018] The present invention overcomes the shortcomings of the prior art and provides a method for introducing a single base into a thio-modified nucleic acid for targeted enrichment of polymorphic sites. Figure 2 As shown, the selector structure is as follows Figure 1 As shown, the selector contains a primer extension binding region of more than 4 bases. When the selector uses the target DNA / RNA as a template, it is extended under the conditions of a single thio-modified dNTP and three other terminating dNTPs / ddNTPs (if the extended base is mutant, then at least two bases will be extended: at least one is thio-modified and one is a terminating dNTP; if the extended base is wild-type, then one base will be extended: one is a terminating dNTP), and after denaturation and renaturation, it will self-form a hairpin structure. Under the action of T4 DNA polymerase, the selector without thio-modification is digested and the sequence at the cut is filled to form a hairpin structure intermediate product, thereby achieving the purpose of enriching rare mutation sites. This technology is particularly suitable for DNA and RNA templates. The obtained hairpin loop structure intermediate can be amplified in two ways: nicking enzyme-mediated nucleic acid amplification or linearization of the loop structure followed by PCR amplification.

[0019] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

[0020] (1) Multiplex PCR or the BDA method based on multiplex PCR is greatly limited in the maximum detection multiplicity. However, the present invention uses a single primer extension reaction. The number of multiplicity that can be enriched in a single tube reaction can refer to the probe hybridization capture technology, and theoretically can reach 10,000 or even tens of thousands of targets;

[0021] (2) The probe capture technology MAESTRO has advantages over the current popular technology, but requires pre-library construction, which will lose some information. The present invention (similar to MAESTRO, both are based on single primer / probe hybridization) can maintain the basis of multiplicity and does not require pre-library construction. In addition, during the enrichment process, multiple rounds of single primer extension can be performed, resulting in dozens of times of enrichment of mutation information.

[0022] (3) The early molecular inversion probe (MIP) genotyping technology required a probe longer than 100 nt (the synthesis cost of each base of the primer synthesis longer than 90 nt is doubled), and MIP requires both ends of the probe to bind to the target at the same time to be effective. However, there is ineffective binding between the probe and the template, such as only one end binding or two different probes binding to the same target (considering that the length of cfDNA is only 160 nt, if the mutation site is located at both ends, then these templates cannot be enriched). More importantly, each round of gap filling in MIP takes 30 minutes, making it difficult to perform multiple rounds of amplification. The present invention requires a synthesized probe of about 70 nt, which only involves the extension of a single-end primer, which can be completed in a few seconds and can be enriched by increasing the number of cycles. The later extension or ligation reaction can be completed in a few minutes. With the method of the present invention, more than 1,000 probes can be multiplexed in one PCR tube using standard laboratory equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the structure of the selector in the method of the present invention;

[0024] Figure 2 1 is a schematic flow chart of the steps for enriching allele information in the method of the present invention;

[0025] Figure 3 is the result of self-extension of the hairpin structure with the last base at the 3' end of DNA modified by thiolation in the embodiment of the present invention; wherein, Figure 3 A: The synthetic template contains 4, 5, or 6 base-paired internal binding primers with a GC content of approximately 40-60% and a phosphorothioate-modified last base. Extension is performed at 25°C in a T4 DNA polymerase reaction system. Figure 3 B: The synthetic template contains 5, 6, or 7 base-paired internal binding primers with a GC content of approximately 0% and a phosphorothioate-modified last base. Extension is performed at 25°C in a T4 DNA polymerase reaction system. Figure 3 C: The synthetic template contains a conventional sequence of three 100% GC base-pairing internal primers HP3GC and four 100% AT base-pairing internal primers HP4AT, and is extended at 25°C in a T4 DNA polymerase reaction system; Figure 3 D, 3E: 4 base-paired internal binding primers with different GC contents and 5 or 6 100% AT internal primers, the last base of which contains a thioate modification, are extended at 25°C in a T4 DNA polymerase reaction system. Figure 3 D. Figure 3 E represents extension at decreasing temperature, and the decreasing temperature is set as: 37°C, 10 minutes, 25°C, 10 minutes, and 15°C, 10 minutes. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] Example 1

[0028] Relationship between stem sequence composition and length and self-extension in synthetic DNA 3'-terminal hairpin structures with thiolation of the last base

[0029] The synthesized primer sequences are shown in Table 1, where * indicates a thiolated modified base and the underlined region is the primer binding region. In a T4 DNA polymerization reaction system containing dNTPs, primer extension was performed at different temperatures, with the "decreasing" temperature used being 37°C for 10 minutes, 25°C for 10 minutes, and 15°C for 10 minutes, to investigate the effect of terminal thiolation on the self-extension of hairpin primers. Figure 3 C is ordinary agarose electrophoresis, and the others are non-denaturing polyacrylamide electrophoresis.

[0030] Table 1 Synthetic DNA sequences

[0031]

[0032]

[0033] Specific methods:

[0034] 1. Template treatment: 90 degrees for 1 minute, then on ice for 2 minutes.

[0035] 2. Reaction system:

[0036] NEBuffer r2.1 1ul, template (10uM) 2ul, T4 DNA polymerase 0.2ul, dNTP, and water to make up to 10ul.

[0037] Reaction conditions 1: 37 degrees for 10 minutes, then 75 degrees for 20 minutes.

[0038] Reaction condition 2: 37 degrees for 10 minutes, 25 degrees for 10 minutes, 15 degrees for 10 minutes, and then 75 degrees for 20 minutes.

[0039] 3. The product is subjected to DNA electrophoresis using polyacrylamide:

[0040] Reagents used:

[0041] Acrylamide solution 45% (100 mL contains 43.4 g of acrylamide and 1.6 g of N, N'-methylenebisacrylamide)

[0042] Ammonium persulfate 0.1g / mL: Weigh 1g of ammonium persulfate and add water to 10mL.

[0043] TBE electrophoresis master solution (5×):

[0044] 1) Weigh 54 g of Tris base, 27.5 g of boric acid, and 20 mL of 0.5 mol / EDTA (pH 8.0).

[0045] 2) Add each group to a 1-liter beaker and adjust the volume to 1 liter with ddH2O.

[0046] 3) Use 1× working solution for electrophoresis and mix it with water at a ratio of 1:4.

[0047] 4) Prepare gel solution (5%): original gel: 8.34 mL, 5xTBE: 15 mL, double distilled water: 6.66 mL, TEMED: 30-36 uL, ASP: 240 uL.

[0048] 5) The product was electrophoresed in a polyacrylamide gel, and then the gel was immersed in 1× Gelred dye for 2 hours and photographed.

[0049] The electrophoresis results are as follows Figure 3 shown. Figure 3 showed that when the last base is phosphorothioate-modified, inner primers as short as 4 bases (containing at least one GC pair) can be extended by T4 DNA polymerase ( Figure 3 A, 3D, 3E), but when the GC content of the paired region is low, a lower temperature is required for efficient extension ( Figure 3 D, HP2A2C* results comparison in 3E); when the inner primer does not contain GC pairs, 5 bases are required for efficient extension ( Figure 3 HP4-7AT* in B~3E); therefore, the ideal internal primer structure is: 3N (when containing GC pairing) + thiolate base or 4N (only AT pairing) + thiolate modified base (Note, according to Figure 1 The thio-modified base shown is a thio-modified dNTP that is extended after the primer binds to the template. To this end, we also examined the effect of T4 DNA polymerase on the non-thio-modified primers HP3GC and HP4AT. The results showed that they were easily digested by the exonuclease activity of T4 DNA polymerase ( Figure 3 C), that is, primers without thiolation modification are easily removed by T4 DNA polymerase.

[0050] Conclusion: This experiment verified Figure 2The feasibility of the technology mentioned above for enriching rare mutations by introducing thiolated modified bases is as follows: when the end of the hairpin primer contains a thiolated modified base, it can inhibit the exolytic activity of T4 DNA polymerase and activate the DNA synthesis activity of T4 DNA polymerase, thereby forming a complete hairpin structure; while short hairpin structure primers with less than 4 bases without the introduction of thiolated modification will be removed by the exolytic activity of T4 DNA polymerase.

[0051] Example 2

[0052] According to the COSMIC database, the BRAF gene in A375 cells has a homozygous p.V600E mutation. The p.V600E (c.1799T>A) mutation site of BRAF in A375 cells (based on the sequence NM_004333.6) was detected by introducing thiolated modified bases.

[0053] The specific method is as follows:

[0054] (1) Extract the genome of A375 and 293T (control group) cells;

[0055] (2) 10 ng of genomic DNA was used as a template and primer F was used for PCR amplification;

[0056] Reaction system (10ul): 10ng DNA, 0.2uM F primer, 1mM thio-modified dATP (Hefei Shitong), 1 unit pfu DNA polymerase (Shanghai Biotechnology), 20mM Tris-HCl (pH 8.8), 10mM (NH4)2SO4, 0.1mg / mL BSA, 10mM KCl, 2mM MgSO4, 0.1% Triton X-100.

[0057] Reaction conditions: pre-denaturation at 94°C for 5 minutes, followed by 50 cycles of (94°C for 5 seconds, 56°C for 10 seconds).

[0058] F primer (the uppercase region is the common primer region, the lowercase region is the specific primer region, the uppercase underlined region is the Nt.BstNBI nicking enzyme recognition sequence, the bold italic bases are mutant bases, and the lowercase italic region is the reverse complementary sequence of the last four bases at the end of the primer):

[0059] 5'-GGTTCGACAGATCCTA GAGTC TctgtCGTGTGCTCTTCCGATCTgtgattttggtctagctacag-3';

[0060] (3) After recovering the reaction product from step (2), denature it at 94°C for 1 minute and place it on ice for 2 minutes. Add 2 units of T4 DNA polymerase and 0.2 mM dNTPs, and extend the DNA at a decreasing temperature of 37°C for 2 minutes, 25°C for 2 minutes, and 15°C for 15 minutes.

[0061] (4) Add 0.2 μL of Nt.BstNBI (NEB) to the product obtained in step (3) and incubate at 37°C for 30 minutes;

[0062] (5) Using UF and UR as primers, 2 μL of the product obtained in step (4) was used as a template for quantitative PCR identification;

[0063] UF: 5'-GGTTCGACAGATCCTAGAGTC-3';

[0064] UR: 5'-AGATCGGAAGAGCACACG-3';

[0065] The quantitative PCR results showed that the CT value of the control group (293T cell group) was greater than 35, while the CT value of the A375-containing group was 25.6, which was equivalent to a difference of 2 to the power of 10 between the two groups. Therefore, the mutation detectable by the present invention was at least at the 1 / 1000 level.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for introducing single-base thiolation-modified nucleic acids for targeted enrichment of polymorphic sites, characterized in that: The method comprises the following steps: (1) selecting a selector with a characteristic structure as a primer, and performing multiple rounds of single primer extension using the nucleic acid sample to be tested as a template under the conditions of a DNA synthesizer and its corresponding buffer and a single thio-modified base; (2) The extension product obtained in step (1) was purified using a DNA purification kit, and then heated for denaturation at 90° C. for 1 minute, and placed on ice for 2 minutes for intramolecular renaturation to form an intermediate nucleic acid molecule with a hairpin structure having a single-stranded region at the 5′ end; (3) T4 DNA polymerase is added to remove the selector without the introduction of the thiolated modified base, and the gap sequence is filled in by the action of T4 DNA polymerase to form a hairpin-shaped intermediate product; (4) using the intermediate product as a template for nucleic acid amplification and subsequent chip or next-generation sequencing analysis.

2. The method according to claim 1, wherein In step (1), a selector with a characteristic structure is selected as a primer, and multiple rounds of single primer extension are performed using the nucleic acid sample to be tested as a template under the conditions of DNA synthesizer and its corresponding buffer, a single thio-modified base, and a mixture of three other terminating dNTPs.

3. The method according to claim 2, wherein The single thio-modified base is selected from one of dATPɑS, dGTPɑS, dCTPɑS, and dTTPɑS.

4. The method according to claim 2, wherein If the method is used to enrich allele information, in step (1), the selector of the characteristic structure consists of four parts, which are: common primer 1 region, which consists of 18 to 30 bases; a base complementary to the mutant base extended by the specific primer; a primer extension binding region, which consists of 4 to 6 bases, and its sequence is in a reverse complementary relationship with the last base at the 3' end of the specific primer; a common primer 2 region, which consists of 18 to 30 bases; a specific primer region for targeted enrichment of SNP site information, which consists of 18 to 30 bases, and the first base extended is the SNP site base.

5. The method according to claim 4, wherein If the method is used to enrich allele information, in step (1), the selector of the characteristic structure consists of four parts, which are: a common primer 1 region consisting of 18 to 25 bases; a base complementary to the mutant base extended by the specific primer; a primer extension binding region consisting of 4 bases, whose sequence is in a reverse complementary relationship with the last base at the 3' end of the specific primer; a common primer 2 region consisting of 18 to 25 bases; a specific primer region for targeted enrichment of SNP site information, consisting of 18 to 25 bases, and the first base extended is the SNP site base.

6. The method according to claim 4, wherein When the method is used to enrich mutant gene information, in step (1), the sequences of common primer region 1 and common primer region 2 are the same in all selectors, which are primer sequences for PCR amplification of mutation site information and are used for high-throughput sequencing template preparation.

7. The method according to claim 1, wherein In step (1), the nucleic acid sample is DNA or RNA, wherein, depending on the nucleic acid sample, the DNA synthesizing enzyme is Taq DNA polymerase that uses DNA as a template or reverse transcriptase that synthesizes DNA using RNA as a template.

Images