Genome walking method based on sawtooth PCR (Polymerase Chain Reaction), kit and application of genome walking method

Abstract

The invention discloses a genome walking method based on sawtooth PCR, a kit and application thereof, and belongs to the technical field of genome walking. The genome walking method comprises the following steps: continuously carrying out three-wheel sawtooth PCR (Polymerase Chain Reaction) amplification; the three random primers form three different combinations according to different use sequences, the three primers in the different combinations are sequentially matched with the specific primers SP1, SP2 and SP3 to form three groups of parallel reactions, unknown flanks of known sequences are obtained based on walking primer mismatch, non-target products generated in the genome walking process are reduced, the amplification efficiency is improved, and the detection sensitivity is improved. And the quantity demanded for walking primers is reduced. According to the method, the influence of the III-type non-target product can be effectively removed through the unique design of the serrated difference of the random primer, the amplification success rate of a complex or high background sample can be increased, the sensitivity, specificity and repeatability of PCR amplification can be remarkably improved, and the amplification difficulty of a long fragment or a high-GC-content fragment is effectively overcome.

Description

Technical Field

[0001] This invention relates to the field of genome walking technology, specifically to a genome walking method, kit, and application based on sawtooth PCR. Background Technology

[0002] Genome walking is a molecular biology method for obtaining unknown regions flanking known DNA sequences. It plays a crucial role, especially when the genetic information of the biological sequence to be analyzed is limited, and holds an important position in molecular biology and related fields. PCR-based genome walking methods dominate due to their simple principles, rapid operation, and high accuracy. Currently reported genome walking techniques can be mainly classified into three categories based on their basic principles: (1) reverse PCR; (2) ligation-mediated PCR; and (3) random priming PCR.

[0003] Both reverse PCR and ligation-mediated PCR require enzyme digestion and ligation of template DNA, which places high demands on template DNA quality and inevitably limits their practical application. Random priming PCR, such as bridging PCR, primer extension refractory PCR (PER-PCR), and stepwise partially overlapping primer-based PCR (SWPOP-PCR), does not rely on template digestion and ligation, offering simplicity and low cost, and is gradually becoming a research hotspot. However, existing random priming PCR inevitably produces three types of non-target molecules during the experiment: (I) non-target products mediated by two specific primers; (II) non-target products mediated by specific primers and random primers; and (III) non-target products mediated entirely by random primers. These non-target products affect the purity of the target fragment, thus affecting the accuracy of the stepping results. Types I and II non-target products, lacking binding sites on the second and third specific primers, cannot be amplified in secondary and tertiary PCR and are diluted in subsequent reactions. Therefore, eliminating the influence of Class III non-target products has become a key focus in the application of random induced PCR. Summary of the Invention

[0004] To address the problems mentioned in the background art, this invention provides a genome walking method, kit, and applications based on serrated PCR. This method, through the unique design of serrated differences in random primers, effectively removes the influence of Class III non-target products. This not only improves the amplification success rate for complex or high-background samples but also significantly enhances the sensitivity, specificity, and reproducibility of PCR amplification, effectively overcoming the difficulties in amplifying long fragments or high-GC-content fragments.

[0005] In this application, a novel random priming PCR-saw-tooth PCR (ST-PCR) method was established, and the feasibility of ST-PCR was further verified by walking the dnaK, pduC, and ALDOA genes.

[0006] To achieve the above objectives, the present invention provides a genome walking method based on sawtooth PCR, comprising the following steps: (1) Primary amplification: Using genomic DNA as a template, hot-start DNA polymerase, random primers, and specific primer SP1 are added to a reaction system containing dNTPs and PCR buffer. Amplification is carried out through annealing temperature graded cycles. The annealing temperature graded cycles consist of three annealing stages: the first stage includes 5 high-rigidity cycles; the second stage includes 1 very low-rigidity cycle; and the third stage includes 25-35 high-rigidity cycles. (2) Secondary amplification: Based on the primary amplification product, random primers and specific primer SP2 are added to the reaction system containing dNTPs and PCR buffer, and amplification is carried out by annealing temperature graded cycles; the annealing temperature graded cycles consist of two annealing stages: the first stage includes one very low-rigidity cycle; the second stage includes 25-35 high-rigidity cycles. (3) Tertiary amplification: Based on the secondary amplification products, random primers and specific primer SP3 are added to the reaction system containing dNTPs and PCR buffer, and amplification is carried out by annealing temperature graded cycles; the annealing temperature graded cycles consist of two annealing stages: the first stage includes one very low-rigidity cycle; the second stage includes 25-35 high-rigidity cycles. SP1, SP2, and SP3 are nested primers designed based on genomic DNA; The random primers are selected from at least three random primer sets with Tm values ​​between 58 and 61 °C, and are not reused in each stage of PCR amplification. They are all single-stranded oligonucleotides with a length of 25 nt, and the 5′ 11 nt and 3′ 3 nt ends of each pair are completely identical, while the remaining base pairs are randomly maintained with a difference of 7 nt.

[0007] Preferably, the primary amplification reaction solution contains 1 μL of genomic DNA (diluted appropriately if necessary), 1×LAPCR Buffer II (Mg2+). 2+ Plus), 0.4 mM each of dNTPs, 0.2 μM random primers, 0.2 μM SP1, 2.5 U LA Taq polymerase, and replenished with ultrapure water to 50 µL; the genomic DNA comprises 10-100 ng L. brevis (Lactobacillus brevis) CD0817 or 100-1000ng human genomic DNA.

[0008] Preferably, the secondary amplification reaction solution contains 1 μL of the primary amplification reaction product (diluted appropriately if necessary), 1×LA PCR Buffer II (Mg 2+ Plus), 0.4 mM each of dNTPs, 0.2 μM random primers, 0.2 μM SP2, and replenished to 50 µL with ultrapure water.

[0009] Preferably, the tertiary amplification reaction solution contains 1 μL of the secondary amplification reaction product (diluted appropriately if necessary), and 1×LA PCR Buffer II (Mg 2+ Plus), 0.4 mM each of dNTPs, 0.2 μM random primers, 0.2 μM SP3, and replenished to 50 µL with ultrapure water.

[0010] Preferably, the random regions of the random primers are sequences with a GC content between 40% and 60%, and there are no severe hairpin structures or dimers between primers (Tm value difference ≤ 3℃).

[0011] Preferably, SP1, SP2, and SP3 are selected from those according to... L. brevis Three specific primer sets with Tm values ​​of 60-65℃ were screened from the DNA sequences of the dnaK gene, pduC gene and ALDOA gene in the human genome. The sequences of the specific primers are shown in SEQ ID NO: 4 to SEQ ID NO: 12 in the sequence listing.

[0012] Preferably, the high-rigidity cycling temperature is 60°C, and the very low-rigidity cycling temperature is 30°C.

[0013] Preferably, during the cycle, the denaturation temperature is 95°C for 30 seconds, and the extension temperature is 72°C for 2 minutes.

[0014] Preferably, the random primer set includes STP1, STP2, and STP3, the sequences of which are shown in SEQ ID NO: 1 to SEQ ID NO: 3 in the sequence listing.

[0015] A second aspect of the present invention provides a genome walking kit for the method of claim 1, comprising: (1) Nested gene-specific primer set: complementary to known genomic DNA sequences, concentration 0.2 μM; (2) Random primer set: 25nt in length, with 11nt at the 5′ end and 3nt at the 3′ end of each pair being identical, and the remaining base pairs maintaining a random 7nt difference, concentration 0.2μM; (3) Taq polymerase: contains anti-inhibitor components; (5) PCR buffer: containing 1.5 mmol / L Mg 2+ 50 mmol / LK + and 10 mmol / L Tris-HCl; (6) dNTP mixture: 0.4 mM concentration of each dNTP.

[0016] Preferably, both the random primer set and the specific primer set are purified by PAGE with a purity ≥95%.

[0017] A third aspect of the present invention provides the application of the method or kit described in the present invention in the fields of molecular biology, genetic engineering or breeding.

[0018] Preferably, the applications include cloning the full-length sequence of the target gene (including regulatory regions such as promoters and terminators), analyzing cis-acting elements flanking the gene (which can also be used to analyze sequence features such as intron-exon boundaries flanking the gene), constructing genome contigs (to complete gap filling and splicing of genome sequencing), identifying insertion sites (T-DNA, transposons, etc.) of inserted mutants (to locate mutant genes), or using molecular marker-assisted breeding (to develop molecular markers based on gene flanking sequences).

[0019] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Simple and efficient. Multiple parallel ST-PCRs can be set up by simply changing the order of random primers. Multiple primer combinations can be tested in one experiment, which greatly increases the probability of finding effective STP binding sites, avoids the randomness of a single attempt, and significantly improves the success rate and experimental efficiency of ST-PCR. Especially when dealing with complex genomic environments and bands with high GC content, the unique random primer serrated structure design makes the genome walking experiment more successful.

[0020] (2) Universality: STP sequences are designed completely randomly, so they are applicable to any genome. In particular, the differential bases in the middle 11nt make STPs cover most possible short sequence combinations. No matter how special the base composition of the target genome is, there are possible effective bindings in STPs.

[0021] (3) High specificity: STP generates a unidirectional extension product under low stringency, and SP performs exponential amplification under high stringency cycling. This process sets a selection threshold, which can only specifically amplify the target fragment that is correctly bound at both ends, effectively filtering out the non-specific background generated by a single STP and obtaining a purer result. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the sawtooth PCR principle in one embodiment of the present invention; Figure 2 This is a graph showing the effect of primary ST-PCR product dilution on secondary PCR in one embodiment of the present invention; Figure 3 This is a graph showing the effect of secondary ST-PCR product dilution on tertiary PCR in one embodiment of the present invention; Figure 4 This is a graph showing the effect of the number of secondary ST-PCR cycles in one embodiment of the present invention; Figure 5 This is a graph showing the effect of the number of cycles in three-stage ST-PCR in one embodiment of the present invention; Figure 6 This is a verification diagram of the uncharacterized genomic regions of three genes in one embodiment of the present invention; Figure 7 This is a graph showing the effect of removing low-strict cycling in the secondary and tertiary PCR processes in the comparative examples of this invention. Figure 8 This is a diagram illustrating the effect of removing the random primer serrated structure in the comparative example of this invention; Figure 9 This is a schematic diagram of the serrated structure of a serrated PCR primer in one embodiment of the present invention. Detailed Implementation

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] 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. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In the following embodiments: 1. Materials and Reagents 1.1 Strains L. brevis The CD0817 strain is deposited at the China Center for Type Culture Collection (CCTCC), with the accession number CCTCC NO: M2018462. 1.2 Equipment and Reagents 1.2.1 Reagents 1.2.2 Equipment 1.3 Experimental Methods 1.3.1 Genomic DNA Extraction The procedure for using the bacterial genomic DNA extraction kit from Shanghai Sangon Biotech Co., Ltd. was followed. L. brevis Genomic DNA was extracted from CD0817. The human genomic DNA was purchased from Conway Century Biotechnology: Human GenomicDNA (CW0565S, CWBIO, Taizhou, China).

[0026] Preparation Example 1: Primer Design PCR primer design aims to obtain gene fragments that can efficiently amplify template DNA sequences. Appropriate primer design is a prerequisite for obtaining high-quality target fragments. In this experiment, the design of specific primers (SPs) conformed to the basic principles of primer design. L. brevis Three nested specific primers (SPs) with Tm values ​​of 60-65℃ were screened from the dnaK gene, pduC gene, and Human genome ALDOA gene of CD0817. Adjacent nested primers should maintain a 50-150 nt interval. In addition, three random primers were designed, each 25 nt in length, with 11 nt at the 5' end and 3 nt at the 3' end, and the remaining 11 nt randomly varying by 7 nt among the three primers. This base difference allows the random primers to form a specific serrated structure during annealing, facilitating the accumulation of the target gene and the removal of Class III non-target products. The primers are shown in Table 1. In the primer design, there were no severe hairpin structures or dimers between the primers. Furthermore, there was no specific order between the random primers; they could be freely combined for three rounds of nested ST-PCR, as shown in Table 2.

[0027] Table 1 List of all primers used in the study Table 1 Primers used in this study Note: The three STPs share the same 5' region (11 nt), 3' region (3 nt), and a serrated, distinct intermediate region. STP: Random primer; SP: Specific primer. Table 2. STP and SP sets in nested PCR Table 2 Pairing of STP permutation with SP set in nested PCRs Note: Each row of 3 STPs is combined with an SP in the same row to perform three parallel ST-PCRs. Each column represents the STP combination.

[0028] like Figure 1 As shown, the technical principle and process of ST-PCR are described using the STP1-STP2-STP3 combination. Note: In the figure, the thin solid line and thin dashed line represent known and unknown sequences, respectively; the arrow and bold line represent primers and primer complements, respectively; SP is a specific primer; STP is a zigzag random primer.

[0029] In primary ST-PCR, five high-rigidity cycles are performed first. During this cycle, pSPs anneal to complementary sites in known regions of the template DNA, amplifying the target single-stranded DNA (ssDNA). A subsequent low-rigidity cycle is performed, where pSTPs anneal randomly to random sites in unknown regions of the ssDNA, amplifying the target band and other bands to their annealing sites. This creates a mixed pool of ssDNA containing both target and non-target bands. Thanks to the serrated differences between STPs, all three STPs can be used as sSTPs in primary ST-PCR, ensuring at least one random primer can anneal to a flanking region, increasing the success rate. Thirty high-rigidity cycles are then performed. In the first high-rigidity cycle, pSPs anneal directionally to the perfect binding sites of the target band's ssDNA, amplifying the double-stranded DNA (dsDNA) defined by pSPs and pSTPs. Subsequent high-rigidity cycles exhibit exponential amplification. Non-target ssDNA, lacking perfect binding sites for any primers, cannot be converted to double-stranded form and is thus diluted.

[0030] In secondary PCR, the first step is a low-rigidity cycle designed to anneal sSTP to unknown regions. There are two annealing methods: one is directional annealing of sSTP to the pSTP binding site, forming a unique serrated structure, which then initiates extension, forming an ssDNA library. The other involves annealing to a more perfect sSTP binding site within the pSTP binding site, resulting in shorter ssDNA. In the subsequent 30 high-rigidity cycles, the target band amplifies exponentially driven by SP and sSTP. Non-target ssDNA, lacking a perfect binding site for any primer, cannot be converted to double-stranded form and is thus diluted.

[0031] The principle and process of tertiary ST-PCR driven by tSP and tSTP are the same as those of secondary ST-PCR, which aims to further eliminate non-target products and ultimately enrich the target molecules into the major products.

[0032] Exploration Example 1: The Effect of Dilution on PCR Different template concentrations have a significant impact on the PCR reaction. Too high a concentration can easily induce template annealing, while too low a concentration is detrimental to the amplification of the target band. This study aimed to investigate the effect of product dilution on the subsequent PCR stage.

[0033] The 50 μL primary reaction system includes 1×LA PCR buffer II. L. brevis Genomic DNA: 10-100 ng, 0.4 mM per dNTP, 0.2 μM per primer, 2.5 U LA Taq polymerase (TaKaRa, Beijing, China). After the primary reaction was completed, the primary PCR products were subsequently diluted according to 0, 10... 1 10 2 10 3 10 4 The sample was diluted several times and used as templates for secondary ST-PCR. The secondary reaction mixture was also 50 μL, including 1×LA PCR buffer II, 0.4 mM per dNTP, 0.2 μM per primer, 1 μL of diluted primary product, and 2.5 U LA Taq polymerase. Electrophoresis results are shown below. Figure 2 As shown, primary products diluted 10-100 times are more suitable for secondary amplification. Note: Lane P represents the primary PCR product; S0, S1, S2, S3, and S4: diluted 0-, 10-, and 10-fold, respectively. 1 10 2 10 3 10 4Secondary ST-PCR products using primary products diluted several times as templates; Lane M: TarakaDL 5000 DNA markers (5000, 3000, 2000, 1500, 1000, 750, 500, 250 and 100 bp).

[0034] In the subsequent tertiary ST-PCR, the effect of secondary ST-PCR product dilution on the tertiary ST-PCR was further analyzed. The secondary ST-PCR product was diluted by 0 and 10%, respectively. 1 10 2 10 3 10 4 The template was doubled for the third-stage ST-PCR. The remaining components of the third-stage reaction were the same as those of the second-stage reaction. Experimental results are as follows: Figure 3 As shown, diluting the product 0-100 times facilitates tertiary ST-PCR amplification. Note: Lane S represents the secondary PCR product; T0, T1, T2, T3, and T4: diluted 0-100 times, 10-100 times respectively. 1 10 2 10 3 10 4 Tertiary ST-PCR products with secondary products diluted several times as templates; Lane M: Taraka DL 5000 DNA markers (5000, 3000, 2000, 1500, 1000, 750, 500, 250 and 100 bp).

[0035] Exploring Example 2: The Effect of Cycle Number on PCR This section first discusses the effect of cycle number on secondary ST-PCR. Total cycle numbers of 10, 20, 30, 40, and 50 were set, with other conditions the same as in Example 1. Electrophoresis results are shown below. Figure 4 As shown, no amplification products appeared when the cycle number was set to 10-20. When the cycle number was set to 30-40, amplification products appeared and the product concentration gradually increased. However, as the cycle number increased, the background also gradually deepened, indicating that non-target products also accumulated in large quantities. Therefore, it is recommended to perform about 30 cycles in secondary ST-PCR. Note: Lane P represents the primary product; S1, S2, S3, S4, and S5: represent the secondary ST-PCR products after 10, 20, 30, 40, and 50 cycles, respectively; Lane M: Taraka DL 5000 DNA markers (5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp).

[0036] Secondary PCR products were used as templates for tertiary ST-PCR, with cycle numbers set to 10, 20, 30, 40, and 50, and other conditions the same as in Experimental Example 1. Electrophoresis results are shown in Figure 5. Similar to secondary ST-PCR, approximately 30 cycles are more suitable for tertiary ST-PCR. Note: Lane S represents the secondary product; T1, T2, T3, T4, and T5 represent tertiary ST-PCR products after 10, 20, 30, 40, and 50 cycles, respectively; Lane M represents Taraka DL 5000 DNA markers (5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp).

[0037] Example 1: PCR reaction at the dnaK site Each genome walking experiment consisted of three parallel ST-PCR sets. Each ST-PCR set consisted of three rounds (primary, secondary, and tertiary) of amplification reactions.

[0038] The 50 μL primary reaction system includes 1×LA PCR buffer II. L. brevis Genomic DNA: 10-100 ng, 0.4 mM per dNTP, 0.2 μM per primer, 2.5 U LA Taq polymerase (TaKaRa, Beijing, China). After the primary reaction, the product was diluted 100-fold as a template for the secondary reaction. The secondary reaction mixture was also 50 μL, including 1×LA PCR Buffer II, 0.4 mM per dNTP, 0.2 μM per primer, 1 μL of diluted primary product, and 2.5 U LA Taq polymerase. After the secondary reaction, the product should also be diluted 100-fold for use in the next reaction. The remaining components of the tertiary reaction were the same as those of the secondary reaction.

[0039] Primary PCR consists of three annealing phases: the first phase has 5 high-strictness (60°C) cycles; the second phase has 1 very low-strictness (30°C) cycle; and the third phase has 30 high-strictness cycles. Secondary PCR begins with one low-strictness cycle (40°C), followed by 30 high-strictness cycles. Detailed thermal cycling parameters for ST-PCR are shown in Table 3.

[0040] Example 2: pduC site PCR reaction Each genome walking experiment consisted of three parallel ST-PCR sets. Each ST-PCR set consisted of three rounds (primary, secondary, and tertiary) of amplification reactions.

[0041] The 50 μL primary reaction system includes 1×LA PCR buffer II, 1 μL of L. brevis CD0817 genomic DNA (10-100 ng), 0.4 mM per dNTP, 0.2 μM per primer, and 2.5 U LA Taq polymerase (TaKaRa, Beijing, China). After the primary reaction, the product is diluted 100-fold as a template for the secondary reaction. The secondary reaction mixture is also 50 μL, including 1×LA PCR buffer II, 0.4 mM per dNTP, 0.2 μM per primer, 1 μL of diluted primary product, and 2.5 U LA Taq polymerase. After the secondary reaction, the product should also be diluted 100-fold for use in the next reaction. The remaining components of the tertiary reaction are the same as those of the secondary reaction.

[0042] Primary PCR consists of three annealing phases: the first phase has 5 high-strictness (60°C) cycles; the second phase has 1 very low-strictness (30°C) cycle; and the third phase has 30 high-strictness cycles. Secondary PCR begins with one low-strictness cycle (40°C), followed by 30 high-strictness cycles. Detailed thermal cycling parameters for ST-PCR are shown in Table 3.

[0043] Example 3: PCR reaction at the ALDOA site Each genome walking experiment consisted of three parallel ST-PCR sets. Each ST-PCR set consisted of three rounds (primary, secondary, and tertiary) of amplification reactions.

[0044] The 50 μL primary reaction system includes 1×LA PCR buffer II, human genomic DNA: 100-1000 ng, 0.4 mM per dNTP, 0.2 μM per primer, and 2.5 U LA Taq polymerase (TaKaRa, Beijing, China). After the primary reaction, the product is diluted 100-fold as a template for the secondary reaction. The secondary reaction mixture is also 50 μL, including 1×LA PCR buffer II, 0.4 mM per dNTP, 0.2 μM per primer, 1 μL of diluted primary product, and 2.5 U LA Taq polymerase. After the secondary reaction, the product should also be diluted 100-fold for use in the next reaction. The remaining components of the tertiary reaction are the same as those of the secondary reaction.

[0045] Primary PCR consists of three annealing phases: the first phase has 5 high-strictness (60°C) cycles; the second phase has 1 very low-strictness (30°C) cycle; and the third phase has 30 high-strictness cycles. Secondary PCR begins with one low-strictness cycle (40°C), followed by 30 high-strictness cycles. Detailed thermal cycling parameters for ST-PCR are shown in Table 3.

[0046] Table 3 ST-PCR thermal cycling parameters Table 3 Thermal cycling parameters of ST-PCR Experimental results of Examples 1-3 PCR product processing and sequencing The PCR products were separated by electrophoresis on a 1.5% agarose gel. The electrophoresis results are as follows: Figure 6 As shown, clear DNA bands are visible. Following the operating procedures of the MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0, the clear DNA bands in the secondary or tertiary PCR products were excised and recovered from the gel. The recovered products were sent to Sangon Biotech Co., Ltd. for direct sequencing. Note: a represents the dnaK gene, b represents the pduC gene, and c represents the ALDOA gene. I, II, and III represent three random primer combinations in ST-PCR, according to Table 2; lane P represents the primary product; S represents the secondary product; T represents the tertiary product; the triangle markers point to the clear primary target band; lane M: Taraka DL5000 DNA markers (5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp); the bands marked by the arrows in the figure were recovered and sequenced. These bands were sequenced, and the sequencing results were aligned with MegAlign. The SP-side regions overlapped with the corresponding known DNA regions, confirming the correctness of the ST-PCR amplification. The longest bands in the walking experiments ranged from 2.0 to 8.0 kb.

[0047] Comparative Example 1: No Low-Strict Cyclic Amplification This comparative example uses the second group of amplification at the dnaK gene locus, with secondary and tertiary PCR cycles set up without low-strict cycles. This genome walking experiment consists of three rounds (primary, secondary, and tertiary) of amplification reactions.

[0048] The 50 μL primary reaction system includes 1×LA PCR buffer II, 1 μL of L. brevis CD0817 genomic DNA (10-100 ng), 0.4 mM per dNTP, 0.2 μM per primer, and 2.5 U LA Taq polymerase (TaKaRa, Beijing, China). After the primary reaction, the product is diluted 100-fold as a template for the secondary reaction. The secondary reaction mixture is also 50 μL, including 1×LA PCR buffer II, 0.4 mM per dNTP, 0.2 μM per primer, 1 μL of diluted primary product, and 2.5 U LA Taq polymerase. After the secondary reaction, the product should also be diluted 100-fold for use in the next reaction. The remaining components of the tertiary reaction are the same as those of the secondary reaction.

[0049] Primary PCR consists of three annealing phases: the first phase has 5 high-strictness (60°C) cycles; the second phase has 1 very low-strictness (30°C) cycle; and the third phase has 30 high-strictness cycles. Secondary PCR is performed directly with 30 high-strictness cycles. Detailed thermal cycling parameters are shown in Table 3.

[0050] The PCR products were separated by electrophoresis on a 1.5% agarose gel. The electrophoresis results are as follows: Figure 7 As shown, compared to the control group that underwent one round of low-rigidity cycling, the tertiary PCR products without low-rigidity cycling exhibited a phenomenon where bright bands could not be separated in the sample wells. Note: Lane P represents the primary product; S1 represents the second round product of secondary ST-PCR without low-rigidity cycling; T1 represents the third round product of tertiary ST-PCR without low-rigidity cycling; S2 represents the second round product of secondary ST-PCR with normal low-rigidity cycling; T2 represents the third round product of tertiary ST-PCR with normal low-rigidity cycling; M represents the DNA 5000 Marker (band sizes from top to bottom are 5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp).

[0051] Comparative Example 2: Amplification of the non-serrated structure This comparative example uses the second group of amplification at the dnaK gene locus, employing a three-round cycle of random primers without serrated structures. All three rounds of genome walking are performed using STP2. This genome walking experiment consists of three rounds (primary, secondary, and tertiary) of amplification reactions.

[0052] The 50 μL primary reaction system includes 1×LA PCR buffer II, 1 μL of L. brevis CD0817 genomic DNA (10-100 ng), 0.4 mM per dNTP, 0.2 μM per primer, and 2.5 U LA Taq polymerase (TaKaRa, Beijing, China). After the primary reaction, the product is diluted 100-fold as a template for the secondary reaction. The secondary reaction mixture is also 50 μL, including 1×LA PCR buffer II, 0.4 mM per dNTP, 0.2 μM per primer, 1 μL of diluted primary product, and 2.5 U LA Taq polymerase. After the secondary reaction, the product should also be diluted 100-fold for use in the next reaction. The remaining components of the tertiary reaction are the same as those of the secondary reaction.

[0053] Primary PCR consists of three annealing phases: the first phase has 5 high-strictness (60°C) cycles; the second phase has 1 very low-strictness (30°C) cycle; and the third phase has 30 high-strictness cycles. Secondary PCR begins with one low-strictness cycle (40°C), followed by 30 high-strictness cycles. Detailed thermal cycling parameters are shown in Table 3.

[0054] The PCR products were separated by electrophoresis on a 1.5% agarose gel. The electrophoresis results are as follows: Figure 8 As shown, compared to the control group using serrated differential step primers, using only a single step primer resulted in multiple faint bands in the secondary PCR. In the tertiary PCR, the bright bands were all within the sample wells, with no target band. Note: Lane P represents the primary product; S1 represents the second-round product of the secondary ST-PCR using a single step primer; T1 represents the third-round product of the tertiary ST-PCR using a single step primer; S2 represents the second-round product of the secondary ST-PCR using serrated differential random primers; T2 represents the third-round product of the tertiary ST-PCR using serrated differential random primers; M represents the DNA 5000 Marker (band sizes from top to bottom are 5000, 3000, 2000, 1500, 1000, 750, 500, 250, and 100 bp).

[0055] analyze This study established a genome walking method based on ST-PCR, aiming to effectively remove class III non-target products. The STP used in primary PCR confirmed the annealing mode of random primers, and the STP used in secondary or tertiary ST-PCR effectively eliminated non-target products. In each ST-PCR cycle, a low-rigidity cycle was first performed to induce partial annealing in the STP to guide the synthesis of new non-target ssDNA, ensuring that in the next high-rigidity cycle, the newly generated ssDNA and its template were perfectly bound to any primer for amplification. This prevented the dilution of non-target products in this round of ST-PCR. The clear bands shown in the secondary and tertiary PCRs in this study were all target bands, further confirming the accuracy and feasibility of ST-PCR.

[0056] like Figure 9 The diagram shown is a schematic diagram of the serrated structure of the serrated PCR primers of this invention.

[0057] In randomized PCR experiments, regardless of the design principle of the random primers, a precise match of at least two nucleotides is required at the 3' end. This precise match is a necessary condition for initiating DNA extension after successful annealing at complementary sites. This study preserved a 3-base overlap at the 3' end in the random primer design to ensure efficient DNA extension. The 11 bp random variation in the middle facilitates personalized annealing of different STPs in primary ST-PCR, ensuring the success rate and efficiency of genome walking. Theoretically, using more than one STP combination should result in at least one positive result, and some may produce satisfactory target bands, achieving the experimental results. The overlapping region at the 5' end ensures the stability of the serrated structure and reduces the concentration of non-target products. The observation of multiple bands in secondary / tertiary ST-PCR is a common occurrence in PCR-based genome walking because the walking primers have multiple possible annealing sites in unknown regions. Furthermore, it is normal for secondary PCR bands to be slightly larger than their corresponding tertiary PCR bands, which is influenced by the positional relationship of nested primers.

[0058] In the low-strictness cycles of secondary / tertiary ST-PCR, random primer annealing occurs in two forms: first, annealing directly at the annealing site of the previous PCR random primer, in which case the product length is not significantly different from the previous round; second, the random primer in this round has a better matching site within the annealing site of the previous round's random primer, thus annealing within the random primer, resulting in a shorter target band compared to the previous round. Internal annealing of random primers is more conducive to the formation of multiple bands and does not affect the specificity of ST-PCR. However, in actual experiments, the band patterns of tertiary ST-PCR are similar to those of the corresponding secondary PCR, indicating that internal annealing is relatively rare.

[0059] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A genome walking method based on sawtooth PCR, characterized in that, Includes the following steps: (1) Primary amplification: Using genomic DNA as a template, hot-start DNA polymerase, random primers, and specific primer SP1 are added to a reaction system containing dNTPs and PCR buffer. Amplification is carried out through annealing temperature graded cycles. The annealing temperature graded cycles consist of three annealing stages: the first stage includes 5 high-rigidity cycles; the second stage includes 1 very low-rigidity cycle; and the third stage includes 25-35 high-rigidity cycles. (2) Secondary amplification: Based on the primary amplification product, random primers and specific primer SP2 are added to the reaction system containing dNTPs and PCR buffer, and amplification is carried out by annealing temperature graded cycles; the annealing temperature graded cycles consist of two annealing stages: the first stage includes one very low-rigidity cycle; the second stage includes 25-35 high-rigidity cycles. (3) Tertiary amplification: Based on the secondary amplification products, random primers and specific primer SP3 are added to the reaction system containing dNTPs and PCR buffer, and amplification is carried out by annealing temperature graded cycles; the annealing temperature graded cycles consist of two annealing stages: the first stage includes one very low-rigidity cycle; the second stage includes 25-35 high-rigidity cycles. SP1, SP2, and SP3 are nested primers designed based on genomic DNA; The random primers are selected from at least three random primer sets with Tm values ​​between 58 and 61 °C, and are not reused in each stage of PCR amplification. They are all single-stranded oligonucleotides with a length of 25 nt, and the 5′ 11 nt and 3′ 3 nt ends of each pair are completely identical, while the remaining base pairs are randomly maintained with a difference of 7 nt.

2. The method according to claim 1, characterized in that, The primary amplification reaction solution contained 1 μL of genomic DNA, 1×LA PCR Buffer II, 0.4 mM of each dNTP, 0.2 μM of random primers, 0.2 μM of SP1, 2.5 U of LA Taq polymerase, and was replenished to 50 μL with ultrapure water; the genomic DNA contained 10-100 ng of... L. brevis CD0817 or 100-1000ng of human genomic DNA; and / or The secondary amplification reaction solution contains 1 μL of primary amplification reaction product, 1×LA PCR Buffer II, 0.4 mM each of dNTPs, 0.2 μM of random primers, 0.2 μM of SP2, and is made up to 50 µL with ultrapure water. and / or The tertiary amplification reaction solution contains 1 μL of the secondary amplification reaction product, 1×LA PCR Buffer II, 0.4 mM each of dNTPs, 0.2 μM of random primers, 0.2 μM of SP3, and is made up to 50 µL with ultrapure water.

3. The method according to claim 1, characterized in that, The random regions of the random primers are sequences with a GC content between 40% and 60%, and there are no severe hairpin structures or dimers between primers.

4. The method according to claim 1, characterized in that, SP1, SP2, and SP3 are selected from according to... L. brevis Three specific primer sets with Tm values ​​of 60-65℃ were screened from the DNA sequences of the dnaK gene, pduC gene and ALDOA gene in the human genome. The sequences of the specific primers are shown in SEQ ID NO: 4 to SEQ ID NO: 12 in the sequence listing.

5. The method according to claim 1, characterized in that, The high-toughness cycling temperature is 60°C, and the very low-toughness cycling temperature is 30°C; and / or During the cycle, the denaturation temperature was 95℃ for 30 seconds, and the extension temperature was 72℃ for 2 minutes.

6. The method according to claim 1, characterized in that, The random primer set includes STP1, STP2, and STP3, and their sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 3 in the sequence listing.

7. A genome walking kit for use in the method of claim 1, characterized in that, include: (1) Nested gene-specific primer set: complementary to known genomic DNA sequences, concentration 0.2 μM; (2) Random primer set: 25nt in length, with 11nt at the 5′ end and 3nt at the 3′ end of each pair being identical, and the remaining base pairs maintaining a random 7nt difference, concentration 0.2μM; (3) Taq polymerase: contains anti-inhibitor components; (5) PCR buffer: containing 1.5 mmol / L Mg 2+ 50 mmol / LK + and 10 mmol / L Tris-HCl; (6) dNTP mixture: 0.4 mM concentration of each dNTP.

8. The reagent kit according to claim 7, characterized in that, Both the random primer set and the specific primer set were purified by PAGE, with a purity ≥95%.

9. The application of the method of claim 1 or the kit of claim 7 in the fields of molecular biology, genetic engineering or breeding.

10. The application according to claim 9, characterized in that, The applications include cloning the full-length sequence of the target gene, analyzing cis-acting elements flanking the gene, constructing genomic contigs, identifying the insertion sites of T-DNA insertion mutants, or using molecular marker-assisted breeding.

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