A sequencing technique for post-library enrichment of mutations

Abstract

This invention discloses a sequencing technique for enriching mutations after library construction. The co-priming amplification (Co-PCR) technique of this invention couples a mutation probe to a short P5 / P7 primer. This short primer has a low melting temperature (Tm) and cannot hybridize and amplify on its own at the reaction temperature. Only when the mutation probe hybridizes to the mutation site can it assist the coupled short P5 / P7 primer in hybridizing to the adjacent P5 / P7 sequence at the end of the pre-library, initiating amplification. Therefore, this Co-PCR technique not only amplifies a specific mutation in a specified gene but also amplifies from both ends of the original pre-library, replicating the complete sequence information of the pre-library, including sequencing adapters, indexes, and sequencing primers. The amplified products can be directly sequenced after purification. By optimizing the co-primers, Co-PCR enrichment amplification of five sites in the pre-library was achieved, resulting in nearly 100-fold enrichment.

Description

Technical Field

[0001] This invention relates to the field of next-generation sequencing, specifically a sequencing technology for enriching mutations after library construction. Background Technology

[0002] Using next-generation sequencing (NGS) to analyze genomic variations in cancer patients has become a major trend in clinical tumor detection. The 2024 consensus report, "Consensus on Molecular Residual Lesion Detection in Solid Tumors," explicitly states that NGS-based ctDNA mutation detection is currently the most commonly used method for detecting MRD in solid tumors. Targeted next-generation sequencing (NGS) enriches genes or gene regions of interest from the whole-genome background, accurately detecting tumor-related mutations and variations without requiring deep sequencing of the entire genome, offering lower cost and higher sensitivity. Targeted sequencing technologies mainly include amplicon sequencing and hybridization capture.

[0003] In hybridization capture methods, genomic DNA is fragmented using enzymatic or acoustic methods, and then adapters and barcode indices are ligated to generate a genomic pre-library library. The pre-library library is then hybridized using biotin-labeled target sequence-specific probes, and streptavidin magnetic beads are used to capture the hybridization probes to isolate the genomic region of interest for sequencing. This method is suitable for targeted sequencing of large-scale genomes, covering large gene regions and comprehensively analyzing all variant types. In liquid biopsy, hybridization capture technology is widely used for ctDNA detection, particularly excelling in TMB and CNV analysis. However, its high target sequence coverage and uniformity are only suitable for large-scale gene capture; the on-target rate (the percentage of sequencing data mapped to the target region) is less than ideal in small panels. In whole-exome sequencing, the target hit rate of hybridization capture sequencing can reach 95%. However, when capturing hundreds of genes, the capture region is nearly 3Mb, but the target hit rate is still less than 50%. For the capture of dozens of genes, the target hit rate is as low as 37%. The fewer the genes captured and the smaller the gene region, the higher the off-target rate. On the other hand, hybridization capture-based targeted sequencing technology requires the design of many capture probes for each site. The arrangement of probes, coverage density, probe length, and the range of the target region all affect the capture. Hybridization time varies from 2 hours to 24 hours, which is time-consuming and labor-intensive. Longer hybridization times can improve the capture effect, but they can also introduce non-specific hybridization. Although both targeted sequencing technologies are more economical than whole-genome sequencing, they essentially only enrich the target gene and reduce the data and cost waste caused by the amplification of other uninteresting genes, but they do not reduce the interference of wild-type fragments of the target gene on mutation amplification. Therefore, the mutation detection sensitivity of current targeted sequencing detection technologies is still limited.

[0004] Therefore, it is necessary to improve such technology to overcome the aforementioned shortcomings. Summary of the Invention

[0005] The purpose of this invention is to provide a sequencing technology for enriching mutations after library construction, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A co-amplification primer targeting a specific mutation is specifically formed by linking an azide-modified Azide probe and a dibenzocyclooctylene (DBCO)-modified DBCO primer via a click chemistry reaction; the DBCO primer includes either a DBCO-modified P5 primer or a DBCO-modified P7 primer; the Azide probe includes either an Azide-modified mutant probe (Azide-Mut) or an R probe (Azide-R) (reverse probe).

[0008] Furthermore, the DBCO-modified P5 primer has a Tm value of 45.5℃, is DBCO-modified at the 5' end, and has the sequence: DBCO-AATGATACGGCG; the DBCO-modified P7 primer has a Tm value of 42.8℃, is DBCO-modified at the 5' end, and has the sequence: DBCO-CAAGCAGAAGAC.

[0009] Furthermore, the Azide-modified mutant probe has a Tm of 55-65℃, a mutation site in the middle of the probe, and may or may not have LNA modification; it is 5' end Azide-modified and 3' end phosphorylated and blocked.

[0010] Furthermore, the Azide-modified R probe and the Azide-modified mutant probe are located on opposite strands of the DNA, respectively, with a Tm of 70°C, Azide azido-modification at the 5' end, phosphorylation blocking at the 3' end, and the sequence is located within 50 bp downstream of the mutation site. The Azide-modified mutant probe and the R probe hybridize to the forward and reverse strands of the DNA, respectively. For five target gene mutations: PIK3CA E545K, EGFR E746_A750del, EGFR T790M, EGFR L858R, and EGFR G719S, Azide-modified mutant probes (Azide-Mut) and R probes (Azide-R) were designed, and their sequence information is shown in Table 1.

[0011] The click chemistry reaction of Azide with DBCO is a copper-strain-free, strain-promoted acetylene-azidocycloaddition (SPAAC) reaction. This reaction is bioorthogonal, requires no metal catalyst, exhibits rapid kinetics and high stability in aqueous buffer, and generates stable triazole bonds.

[0012] Synthesized coprimers for co-amplification via copper-free click chemistry, including:

[0013] Azide probe:DBCO primer = 1:2, i.e., final concentrations of 10 μM and 20 μM respectively. React in 0.7 x PBS at 37°C for 4 h. Store at -20°C. The final coprimer concentration is calculated based on the Azide probe concentration, i.e., 10 μM.

[0014] like Figure 1 As shown, the synthesis method of the coprimer Mut-P5 / P7 is as follows:

[0015] Click on chemical reaction 1: Azide-Mut + DBCO-P7 = Mut-P7 coprimer

[0016] Click on chemical reaction 2: Azide-Mut + DBCO-P5 = Mut-P5 coprimer

[0017] like Figure 2 As shown, the synthesis method of the coprimer R-P5 / P7 is as follows:

[0018] Click on chemical reaction 3: Azide-R+ DBCO-P7 = R-P7 coprimer

[0019] Click on chemical reaction 4: Azide-R+ DBCO-P5 = R-P5 coprimer

[0020] Wherein, Azide-Mut represents an Azide-modified mutant probe; Azide-R represents an Azide-modified R probe; DBCO-P7 represents a DBCO-modified P7 primer; and DBCO-P5 represents a DBCO-modified P5 primer.

[0021] The application process of Co-PCR mutant enrichment technology in NGS is as follows: Figure 3 This includes the following steps:

[0022] S1. Pretext Sample Processing

[0023] The pre-library sample, which had already undergone fragmentation and ligation sequencing adapter processing, was obtained from Geneplus Technology Co., Ltd., with adapter type IDT384, adapter number 248, and a concentration of 50 ng / μL. This sample was diluted 20-fold before being used in subsequent amplification experiments.

[0024] S2. Prepare a five-component synergistic primer mixture.

[0025] According to claim 5, five Mut-P7 coprimer, Mut-P5 coprimer, R-P7 coprimer and R-P5 coprimer were synthesized for five target gene mutations (PIK3CA E545K, EGFR E746_A750del, EGFR T790M, EGFR L858R, EGFR G719S), respectively. The coordinating primers for the five mutation sites were mixed in equal proportions to obtain a mixture of Mut-P7 coprimer, a mixture of Mut-P5 coprimer, a mixture of R-P7 coprimer and a mixture of R-P5 coprimer.

[0026] S3. Prepare the Co-PCR amplification reaction system, with multiple reactions prepared in 10% excess:

[0027] Forward Co-PCR amplification reaction system reagents Added amount Final concentration 2×Robustart Premix-UNG (Probe qPCR) 10 μL 1× Mut-P5 coprimer mixture (10 μM each) 0.4 μL*5 200 nM each R-P7 coprimer mixture (10 μM each) 0.4 μL*5 200 nM each Pretext library sample (2.5 ng / μL) 1 μL 0.125 ng / μL Deionized water Add to 20 μL

[0028] Reverse Co-PCR amplification reaction system reagents Added amount Final concentration 2×Robustart Premix-UNG (Probe qPCR) 10 μL 1× Mut-P7 coprimer mixture (10 μM each) 0.4 μL*5 200 nM each R-P5 coprimer mixture (10 μM each) 0.4 μL*5 200 nM each Pretext library sample (2.5 ng / μL) 1 μL 0.125 ng / μL Deionized water Add to 20 μL

[0029] Add 20 μL of the above reaction mixture to a 200 μL centrifuge tube, and perform the reaction in a PCR instrument according to the following touchdown procedure:

[0030] 50℃ for 2 minutes, 95℃ for 3 minutes

[0031] 3 cycles: 95℃ 10s, 65℃ 2min;

[0032] 3 cycles: 95℃ 10s, 64℃ 2min;

[0033] 3 cycles: 95℃ 10s, 63℃ 2min;

[0034] 3 cycles: 95℃ 10s, 62℃ 2min;

[0035] 3 cycles: 95℃ 10s, 61℃ 2min;

[0036] 3 cycles: 95℃ 10s, 60℃ 2min;

[0037] 3 cycles: 95℃ 10s, 59℃ 2min;

[0038] 3 cycles: 95℃ 10s, 58℃ 2min;

[0039] 3 cycles: 95℃ 10s, 57℃ 2min;

[0040] 15 cycles: 95℃ 10s, 56℃ 2min.

[0041] S4. After mixing the forward Co-PCR amplification product and the reverse Co-PCR amplification product, perform routine NGS library preparation procedures, namely magnetic bead purification, Qubit quantification, capillary electrophoresis quality control, and sequencing. The results are as follows. Figure 4 As shown in Table 2, the mutation rate of the five sites in the initial pre-library sample was 0.5%-5%. After Co-PCR enrichment, the mutation rate reached 75%-100%, which is nearly 100-fold enriched, and the total target rate was high, reaching 69.48%.

[0042] Compared with the prior art, the beneficial effects of the present invention are:

[0043] Co-PCR technology can not only enrich mutations, but also retain sequencing adapters and index information in the pre-library, so that the enriched products can be directly used for NGS sequencing.

[0044] Co-PCR technology demonstrated high specificity and enrichment capacity in multisite enrichment, with the mutation rate of pre-library samples with a mutation rate of about 1% approaching 100% after enrichment. Figure 4 ).

[0045] Targeted sequencing of the small panel (5-gene) achieved a mid-target rate of up to 69.48% (Table 2), overcoming the limitation of low mid-target rate in hybrid capture targeted sequencing in small panel applications, and has the potential to replace traditional hybrid capture targeted sequencing.

[0046] Co-PCR technology replaces multiple steps in hybridization capture sequencing, such as probe hybridization, washing, elution, and amplification, with a one-step selective amplification procedure, greatly simplifying the experimental process and improving detection efficiency. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the synthesis of the co-amplification primer (Mut-P5 / P7 coprimer).

[0048] Figure 2 This is a schematic diagram of the synthesis of the co-amplification primer (R-P5 / P7 coprimer).

[0049] Figure 3 This describes the application process of Co-PCR mutation enrichment technology in NGS.

[0050] Figure 4 The results are obtained by NGS sequencing after enrichment of the five-fold mutation sites by Co-PCR. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0052] A coprimer for specific mutations is formed by linking an azide-modified Azide probe with a DBCO-modified dibenzocyclooctylene primer via a click chemical reaction.

[0053] The DBCO probe includes either a DBCO-modified P5 primer or a DBCO-modified P7 primer.

[0054] Among them, the DBCO-modified P5 primer (DBCO-P5) has a Tm value of 45.5℃, is DBCO-modified at the 5' end, and has the sequence: DBCO-AATGATACGGCG.

[0055] DBCO-modified P7 primer (DBCO-P7): Tm value 42.8℃, 5' end DBCO modified, sequence: DBCO-CAAGCAGAAGAC.

[0056] The Azide probe includes either a mutation probe (Azide-Mut) or an R probe (Azide-R);

[0057] Among them, the Azide-modified mutant probe (Azide-Mut) has a Tm of 55-65℃, the mutation site is in the middle of the probe, and there may or may not be LNA modification; the 5' end is Azide modified, and the 3' end is phosphorylated to block it.

[0058] Azide-modified R probe (Azide-R): This probe and the Azide-modified mutant probe are located on opposite strands of the DNA, respectively. The temperature range (Tm) is 70°C. The 5' end is modified with Azide, and the 3' end is phosphorylated and blocked. The sequence is located within 50 bp downstream of the mutation site. The Azide-modified mutant probe and the R probe hybridize to the forward and reverse strands of the DNA, respectively.

[0059] The DBCO-P7 primer is a truncated P7 primer modified with 5'-terminus dibenzocyclooctyne (DBCO), and its hybridization Tm value is 42.8℃.

[0060] The DBCO-P5 primer is a truncated P5 primer modified with 5'-terminus dibenzocyclooctyne (DBCO), and its hybridization Tm value is 45.5℃.

[0061] The Azide-Mut probe has an azide-modified 5' end and a phosphorylation-modified 3' end to block elongation. The probe can identify wild-type and mutant genes and selectively hybridize mutant genes. Its Tm value is 55-65℃.

[0062] The Azide-R probe has an azide-modified 5' end and a phosphorylation-modified 3' end to block elongation. The probe hybridizes downstream of the mutation site, and its Tm value is 70°C.

[0063] The DBCO-P7 / DBCO-P5 primers and the Azide-Mut / Azide-R probe are reacted via click chemistry to obtain co-amplification primers targeting specific mutations.

[0064] Specifically, the two probes undergo efficient click chemistry with the modified DBCO group and the Azide group to form a stable covalent link.

[0065] The click chemistry reaction of Azide with DBCO is a copper-strain-free, strain-promoted acetylene-azidocycloaddition (SPAAC) reaction. This reaction is bioorthogonal, requires no metal catalyst, exhibits rapid kinetics and high stability in aqueous buffer, and generates stable triazole bonds.

[0066] Synthesized co-amplification primers via copper-free click chemistry reactions, including:

[0067] Azide probe:DBCO primer = 1:2, i.e., final concentrations of 10 μM and 20 μM respectively. React in 0.7 x PBS at 37°C for 4 h. Store at -20°C. The final coprimer concentration is calculated based on the Azide probe concentration, i.e., 10 μM.

[0068] like Figure 1 As shown, the synthesis method of the coprimer Mut-P5 / P7 is as follows:

[0069] Click on chemical reaction 1: Azide-Mut + DBCO-P7 = Mut-P7 coprimer

[0070] Click on chemical reaction 2: Azide-Mut + DBCO-P5 = Mut-P5 coprimer

[0071] like Figure 2 As shown, the synthesis method of the coprimer R-P5 / P7 is as follows:

[0072] Click on chemical reaction 3: Azide-R+ DBCO-P7 = R-P7 coprimer

[0073] Click on chemical reaction 4: Azide-R+ DBCO-P5 = R-P5 coprimer

[0074] Wherein, Azide-Mut represents an Azide-modified mutant probe; Azide-R represents an Azide-modified R probe; DBCO-P7 represents a DBCO-modified P7 primer; and DBCO-P5 represents a DBCO-modified P5 primer.

[0075] Considering the randomness of P5 / P7 sequence ligation during pre-library construction, there may be mutation sites on the same DNA strand as P5 and P7' (the complementary sequence of P7), and there may also be mutation sites on the same DNA strand as P7 and P5' (the complementary sequence of P5).

[0076] When the mutation site is located on the same DNA strand as P5 and P7' (the complementary sequence of P7), the reacted Mut-P7 coprimer hybridizes and anchors to the mutant library fragment, guiding the truncated P7 primer to bind to the P7' sequence at the end of the mutant library fragment, thereby initiating P7 end amplification. To ensure bidirectional amplification of the mutant library fragment, the R-P5 coprimer hybridizes and anchors to the other strand of the library DNA, guiding the truncated P5 primer to bind to the P5' sequence at the end of the mutant library fragment, thereby initiating P5 end amplification. Unlike the Mut-P7 coprimer, the R-P5 coprimer anchors not to the mutation site, but to a downstream sequence of the mutation. It does not distinguish between wild-type and wild-type mutations, and is only used to amplify the gene. Furthermore, the Mut-P7 coprimer and R-P5 coprimer hybridize to the two strands of the library DNA, respectively, acting as the forward and reverse primers in PCR.

[0077] Simultaneous addition of Mut-P7 coprimer and R-P5 coprimer ensures that the library fragment containing the specified mutation of the specified gene can be amplified from both ends of P5 / P7 simultaneously, thus completing the amplification of the target library sequence.

[0078] When the mutation site is located on the same DNA strand as P7 and P5' (the complementary sequence of P5), the reacted Mut-P5 coprimer hybridizes and anchors to the mutant library fragment, guiding the truncated P5 primer to bind to the P5' sequence at the end of the mutant library fragment, thereby initiating P5 end amplification. To ensure bidirectional amplification of the mutant library fragment, the R-P7 coprimer hybridizes and anchors to the other strand of the library DNA, guiding the truncated P7 primer to bind to the P7' sequence at the end of the mutant library fragment, thereby initiating P7 end amplification. Unlike the Mut-P5 coprimer, the R-P7 coprimer anchors not to the mutation site, but to a downstream sequence of the mutation. It does not distinguish between wild-type and wild-type mutations, and is only used to amplify the gene. Furthermore, the Mut-P5 coprimer and R-P7 coprimer hybridize to the two strands of the library DNA respectively, acting as the forward and reverse primers in PCR.

[0079] Simultaneous addition of Mut-P5 coprimer and R-P7 coprimer ensures that the library fragment containing the specified mutation of the specified gene can be amplified from both P5 / P7 coprimers simultaneously, thus completing the amplification of the target library sequence.

[0080] For other genes, or wild-type fragments of the target gene, the Azide-Mut probe cannot hybridize with other genes or wild-type fragments of the target gene. Therefore, it cannot guide the truncated P7 / P5 primer (DBCO-P7 / P5) to amplify the target fragment. Furthermore, the independent DBCO-P7 / P5 primer cannot hybridize to the target fragment to initiate amplification at the reaction temperature due to its low Tm value, resulting in amplification failure.

[0081] The advantage of coprimer lies in its ability to couple the Azide-Mut probe to the DBCO-P5 / P7 primers via click chemistry. The hybridization of the Azide-Mut probe allows the linked DBCO-P5 / P7 primers to get closer to the target gene, thereby increasing the probability of the DBCO-P5 / P7 primers colliding with the P5` / P7` (complementary sequences of P5 / P7) at the end of the target fragment. Even with a low Tm value, it can promote hybridization and initiate amplification.

[0082] Due to the randomness of the pre-library construction process, the target mutation site may be linked to either the P7 primer or the P5 primer. Therefore, in two separate reactions, Mut-P7 coprimer combined with R-P5 coprimer and Mut-P5 coprimer combined with R-P7 coprimer are performed respectively. After the reactions are combined, the target mutation library fragment can be amplified and enriched to the greatest extent.

[0083] Methods for synthesizing co-amplification primers include:

[0084] Azide probe:DBCO primer = 1:2, i.e., final concentrations of 10 μM and 20 μM respectively. React in 0.7 x PBS at 37°C for 4 h. Store at -20°C. The final coprimer concentration is calculated based on the Azide probe concentration, i.e., 10 μM.

[0085] like Figure 1 As shown, the synthesis method of the coprimer Mut-P5 / P7 is as follows:

[0086] Click on chemical reaction 1: Azide-Mut + DBCO-P7 = Mut-P7 coprimer

[0087] Click on chemical reaction 2: Azide-Mut + DBCO-P5 = Mut-P5 coprimer

[0088] like Figure 2 As shown, the synthesis method of the coprimer R-P5 / P7 is as follows:

[0089] Click on chemical reaction 3: Azide-R+ DBCO-P7 = R-P7 coprimer

[0090] Click on chemical reaction 4: Azide-R+ DBCO-P5 = R-P5 coprimer

[0091] Azide-Mut: Azide-modified mutant probe; Azide-R: Azide-modified R probe; DBCO-P7: DBCO-modified P7 primer; DBCO-P5: DBCO-modified P5 primer.

[0092] Co-PCR amplification reaction includes the following steps:

[0093] S1. Pretext Sample Processing

[0094] The pre-library sample, which had already undergone fragmentation and ligation sequencing adapter processing, was obtained from Geneplus Technology Co., Ltd., with adapter type IDT384, adapter number 248, and a concentration of 50 ng / μL. This sample was diluted 20-fold before being used in subsequent amplification experiments.

[0095] S2. Prepare a five-component synergistic primer mixture.

[0096] According to claim 5, five Mut-P7 coprimer, Mut-P5 coprimer, R-P7 coprimer and R-P5 coprimer were synthesized for five target gene mutations (PIK3CA E545K, EGFR E746_A750del, EGFR T790M, EGFR L858R, EGFR G719S), respectively. The coordinating primers for the five mutation sites were mixed in equal proportions to obtain a mixture of Mut-P7 coprimer, a mixture of Mut-P5 coprimer, a mixture of R-P7 coprimer and a mixture of R-P5 coprimer.

[0097] S3. Prepare the Co-PCR amplification reaction system, with multiple reactions prepared in 10% excess:

[0098] Forward Co-PCR amplification reaction system reagents Added amount Final concentration 2×Robustart Premix-UNG (Probe qPCR) 10 μL 1× Mut-P5 coprimer mixture (10 μM each) 0.4 μL*5 200 nM each R-P7 coprimer mixture (10 μM each) 0.4 μL*5 200 nM each Pretext library sample (2.5 ng / μL) 1 μL 0.125 ng / μL Deionized water Add to 20 μL

[0099] Reverse Co-PCR amplification reaction system reagents Added amount Final concentration 2×Robustart Premix-UNG (Probe qPCR) 10 μL 1× Mut-P7 coprimer mixture (10 μM each) 0.4 μL*5 200 nM each R-P5 coprimer mixture (10 μM each) 0.4 μL*5 200 nM each Pretext library sample (2.5 ng / μL) 1 μL 0.125 ng / μL Deionized water Add to 20 μL

[0100] Add 20 μL of the above reaction mixture to a 200 μL centrifuge tube, and perform the reaction in a PCR instrument according to the following touchdown procedure:

[0101] 50℃ for 2 minutes, 95℃ for 3 minutes

[0102] 3 cycles: 95℃ 10s, 65℃ 2min;

[0103] 3 cycles: 95℃ 10s, 64℃ 2min;

[0104] 3 cycles: 95℃ 10s, 63℃ 2min;

[0105] 3 cycles: 95℃ 10s, 62℃ 2min;

[0106] 3 cycles: 95℃ 10s, 61℃ 2min;

[0107] 3 cycles: 95℃ 10s, 60℃ 2min;

[0108] 3 cycles: 95℃ 10s, 59℃ 2min;

[0109] 3 cycles: 95℃ 10s, 58℃ 2min;

[0110] 3 cycles: 95℃ 10s, 57℃ 2min;

[0111] 15 cycles: 95℃ 10s, 56℃ 2min.

[0112] S4. After mixing the forward Co-PCR amplification product and the reverse Co-PCR amplification product, perform routine NGS library preparation procedures, namely magnetic bead purification, Qubit quantification, capillary electrophoresis quality control, and sequencing. The results are as follows. Figure 4 As shown in Table 2, the mutation rate of the five sites in the initial pre-library sample was 0.5%-5%. After Co-PCR enrichment, the mutation rate reached 75%-100%, which is nearly 100-fold enriched, and the total target rate was high, reaching 69.48%.

[0113] Table 1: Sequence information of 5 sites

[0114] "+" indicates LNA modification, and "p" indicates phosphorylation modification.

[0115] Table 2. Results of NGS sequencing detection after co-PCR enrichment of the five-fold mutation.

[0116] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," "outer," "left," and "right," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of this invention is in use, or the orientation or positional relationship commonly understood by those skilled in the art. They are used only for the convenience of describing this invention and for simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

Claims

1. A co-amplification primer targeting a specific mutation, characterized in that, Specifically, it is formed by linking an azide-modified Azide probe with a dibenzocyclooctylene-modified DBCO primer via a click chemistry reaction; the DBCO primer includes either a DBCO-modified P5 primer or a DBCO-modified P7 primer; the Azide probe includes either an Azide-modified mutant probe or an R probe.

2. The co-amplification primer for a specific mutation according to claim 1, characterized in that, The DBCO-modified P5 primer has a Tm value of 45.5℃, is DBCO-modified at the 5' end, and has the sequence: DBCO-CAAGCAGAAGAC; the DBCO-modified P7 primer has a Tm value of 42.8℃, is DBCO-modified at the 5' end, and has the sequence: DBCO-AATGATACGGCG.

3. The co-amplification primer for a specific mutation according to claim 1, characterized in that, The Azide-modified mutant probes have the following characteristics: Tm is 55-65℃, the mutation site is in the middle of the probe, and LNA modification may or may not be present; the 5' end is Azide-modified, and the 3' end is phosphorylated and blocked; Azide-modified mutant probes were designed for five target gene mutations: PIK3CA E545K, EGFR E746_A750del, EGFRRT790M, EGFR L858R, and EGFR G719S.

4. The co-amplification primer for a specific mutation according to claim 1, characterized in that, The Azide-modified R probe has a Tm of 70℃, is 5' end Azide-modified, 3' end phosphorylated and blocked, and its sequence is located within 50 bp downstream of the mutation site. The Azide-modified mutant probe and the R probe hybridize on the forward and reverse strands of the DNA, respectively. Azide-modified R probes were designed for five target gene mutations: PIK3CA E545K, EGFR E746_A750del, EGFR T790M, EGFR L858R, and EGFR G719S.

5. A method for synthesizing coprimer coprimers via copper-free click chemistry, characterized in that, include: Azide probe: DBCO primer = 1:2, i.e., final concentrations of 10 μM and 20 μM respectively. React in 0.7 x PBS at 37℃ for 4 h, and store at -20℃. The final concentration of coprimer is calculated according to the concentration of Azide probe, i.e., 10 μM. The synthesis method of the co-primer Mut-P5 / P7 is as follows: Click on chemical reaction 1: Azide-Mut + DBCO-P7 = Mut-P7 coprimer Click on chemical reaction 2: Azide-Mut + DBCO-P5 = Mut-P5 coprimer The synthesis method of the coprimer R-P5 / P7 is as follows: Click on chemical reaction 3: Azide-R+ DBCO-P7 = R-P7 coprimer Click on chemical reaction 4: Azide-R+ DBCO-P5 = R-P5 coprimer Azide-Mut: Azide-modified mutant probe; Azide-R: Azide-modified R probe; DBCO-P7: DBCO-modified P7 primer; DBCO-P5: DBCO-modified P5 primer.

6. The application procedure of Co-PCR mutant enrichment technology in NGS, characterized in that, Includes the following steps: S1. Pretext Sample Processing The pre-library sample, which had been fragmented and ligated to sequencing adapters, was obtained from Geneplus Technology. The adapter type was IDT384, adapter number was 248, and the concentration was 50 ng / μL. The sample was diluted 20-fold and used for subsequent amplification experiments. S2. Prepare a five-component synergistic primer mixture. Five coprimers (Mut-P7, Mut-P5, R-P7, and R-P5) were synthesized for five target gene mutations: PIK3CA E545K, EGFR E746_A750del, EGFR T790M, EGFR L858R, and EGFR G719S. The coprimers for the five mutation sites were mixed in equal proportions to obtain mixtures of Mut-P7, Mut-P5, R-P7, and R-P5. S3. Prepare the Co-PCR amplification reaction system, with multiple reactions prepared in 10% excess: Forward Co-PCR amplification reaction system and reverse Co-PCR amplification reaction system Add 20 μL of the above reaction mixture to a 200 μL centrifuge tube, and perform the reaction in a PCR instrument according to the following touchdown procedure: 50℃ for 2 minutes, 95℃ for 3 minutes 3 cycles: 95℃ 10s, 65℃ 2min; 3 cycles: 95℃ 10s, 64℃ 2min; 3 cycles: 95℃ 10s, 63℃ 2min; 3 cycles: 95℃ 10s, 62℃ 2min; 3 cycles: 95℃ 10s, 61℃ 2min; 3 cycles: 95℃ 10s, 60℃ 2min; 3 cycles: 95℃ 10s, 59℃ 2min; 3 cycles: 95℃ 10s, 58℃ 2min; 3 cycles: 95℃ 10s, 57℃ 2min; 15 cycles: 95℃ 10s, 56℃ 2min; S4. After mixing the forward Co-PCR amplification product and the reverse Co-PCR amplification product, perform routine NGS library construction operations, namely magnetic bead purification, Qubit quantification, capillary electrophoresis quality control, and sequencing. The results are shown in Figure 4 and Table 2. The mutation rate of the five sites in the initial pre-library sample was 0.5%-5%. After Co-PCR enrichment, the mutation rate reached 75%-100%, which is nearly 100-fold enriched, and the total target rate was high, reaching 69.48%.

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