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CN-122012490-A - Genome walking method based on sawtooth PCR, kit and application thereof

CN122012490ACN 122012490 ACN122012490 ACN 122012490ACN-122012490-A

Abstract

The invention discloses a genome walking method based on sawtooth PCR, a kit and application thereof, belonging to the technical field of genome walking. The genome walking method comprises the steps of continuously carrying out three rounds of saw tooth PCR amplification, forming three different combinations of 3 random primers according to different use sequences, sequentially matching the three primers in the different combinations with specific primers SP1, SP2 and SP3 to form three groups of parallel reactions, obtaining unknown flanks of known sequences based on mismatch of the walking primers, reducing non-target products generated in the genome walking process, improving the amplification efficiency and reducing the demand on the walking primers. The method can effectively remove the influence of class III non-target products through the unique design of the random primer serrated difference, can not only improve the amplification success rate of complex or high background samples, but also remarkably improve the sensitivity, specificity and repeatability of PCR amplification, and effectively overcome the amplification difficulty of long fragments or fragments with high GC content.

Inventors

  • GAO DANDAN
  • TANG QINGCHUN
  • LI HAIXING

Assignees

  • 西北民族大学

Dates

Publication Date
20260512
Application Date
20260126

Claims (10)

  1. 1. A genome walking method based on zigzag PCR, comprising the steps of: (1) The primary amplification, namely adding a hot start DNA polymerase, a random primer and a specific primer SP1 into a reaction system containing dNTPs and a PCR buffer solution by using a annealing temperature grading cycle for amplification, wherein the annealing temperature grading cycle comprises three annealing stages, namely a first stage comprises 5 high-stringency cycles, a second stage comprises 1 extremely low-stringency cycle and a third stage comprises 25-35 high-stringency cycles; (2) The secondary amplification is carried out by adding random primer and specific primer SP2 into a reaction system containing dNTPs and PCR buffer based on the primary amplification product and carrying out amplification through annealing temperature grading circulation, wherein the annealing temperature grading circulation consists of two annealing stages, namely a first stage comprises 1 very low-stringency circulation and a second stage comprises 25-35 high-stringency circulation; (3) The third-stage amplification, namely adding a random primer and a specific primer SP3 into a reaction system containing dNTPs and a PCR buffer solution based on a second-stage amplification product, and carrying out amplification through an annealing temperature grading cycle, wherein the annealing temperature grading cycle consists of two annealing stages, namely a first stage comprises 1 very low-stringency cycle, and a second stage comprises 25-35 high-stringency cycles; the SP1, the SP2 and the SP3 are nested primers designed based on genome DNA; the random primer is selected from a random primer group at least comprising 3 pieces of primers with Tm value of 58-61 ℃, the amplification is not repeated in each PCR, the primers are single-stranded oligonucleotides with the length of 25nt, the 5 'ends 11nt and 3' ends 3nt between every two are completely identical, and the rest base pairs randomly keep the difference of 7 nt.
  2. 2. The method of claim 1, wherein the step of determining the position of the substrate comprises, The primary amplification reaction solution contains 1. Mu.L of genomic DNA, 0.4mM each of 1 XLA PCR BufferII and dNTPs, 0.2. Mu.M of random primer, 0.2. Mu.M of SP1,2.5U of LA Taq polymerase and supplemented to 50. Mu.L with ultrapure water, wherein the genomic DNA comprises 10-100ng L. brevisCD0817 or 100-1000ng of human genomic DNA; And/or The secondary amplification reaction solution contained 1. Mu.L of the primary amplification reaction product, 1 XLA PCR buffer II, 0.4mM each of dNTPs, 0.2. Mu.M of random primer, 0.2. Mu.M of SP2, and was supplemented to 50. Mu.L with ultrapure water; And/or The tertiary amplification reaction solution contained 1. Mu.L of the secondary amplification reaction product, 1 XLA PCR BufferII, 0.4mM each of dNTPs, 0.2. Mu.M of random primer, 0.2. Mu.M of SP3, and was supplemented to 50. Mu.L with ultrapure water.
  3. 3. The method of claim 1, wherein the random region of the random primer is a sequence having a GC content of between 40-60% and no severe hairpin structure or dimer is present from primer to primer.
  4. 4. The method according to claim 1, wherein the SP1, SP2 and SP3 are selected from the group consisting of a dnaK gene, pduC gene of L.brevis CD0817 and a specific primer group with 3 Tm value of 60-65 ℃ respectively selected from the DNA sequences of ALDOA genes in human genome, and the sequences of the specific primers are shown as SEQ ID NOs 4-12 in a sequence table.
  5. 5. The method of claim 1, wherein the step of determining the position of the substrate comprises, A high stringency cycling temperature of 60℃and a very low stringency cycling temperature of 30℃and/or In the cycle, the denaturation temperature was 95℃for 30 s% and the extension temperature was 72℃for 2min.
  6. 6. The method of claim 1, wherein the random primer set comprises STP1, STP2 and STP3, and the sequence of the random primer set is shown as SEQ ID NO: 1-SEQ ID NO:3 in a sequence table.
  7. 7. A genome walking kit for use in the method of claim 1, comprising: (1) Nested gene specific primer group, complementary to the known genome DNA sequence, concentration 0.2 mu M; (2) The length of the random primer group is 25nt, the 5 'end 11nt and the 3' end 3nt between every two of the random primer groups are completely the same, and the difference of 7nt is randomly kept by the rest base pairs, and the concentration is 0.2 mu M; (3) Taq polymerase containing anti-inhibitor component; (5) PCR buffer containing 1.5mmol/L Mg 2+ 、50mmol/L K + and 10mmol/L LTris-HCl; (6) dNTPs mixture, 0.4mM each dNTP concentration.
  8. 8. The kit of claim 7, wherein the random primer set and the specific primer set are purified by PAGE and have a purity of 95% or more.
  9. 9. Use of the method of claim 1 or the kit of claim 7 in the fields of molecular biology, genetic engineering or breeding.
  10. 10. The use according to claim 9, characterized in that it comprises cloning the full-length sequence of the gene of interest, analyzing cis-acting elements flanking the gene, constructing a genome contig, identifying the insertion site of a T-DNA insertion mutant or for molecular marker assisted breeding.

Description

Genome walking method based on sawtooth PCR, kit and application thereof Technical Field The invention relates to the technical field of genome walking, in particular to a genome walking method based on sawtooth PCR, a kit and application thereof. Background Genomic walking techniques are molecular biological methods to obtain unknown regions flanking a known DNA sequence. In particular, genome walking plays an important role when genetic information of biological sequences to be analyzed is limited, and plays an important role in molecular biology and related fields. The genome walking method based on PCR takes the main position due to the characteristics of simple principle, rapid operation, high accuracy and the like. The genome walking technology reported at present can be mainly classified into three types of (1) inverse PCR, (2) ligation-mediated PCR, and (3) random priming PCR according to its basic principle. Both inverse PCR and ligation-mediated PCR require digestion and ligation of template DNA, and have high requirements for template DNA quality, which are inevitably limited in practical applications. And random priming PCR such as bypass PCR (Bridging PCR), primer extension block PCR (Primer Extension Refractory PCR, PER-PCR), primer stepped partial overlap PCR (STEPWISE PARTIALLY overlapping primer-based PCR, SWPOP-PCR) and the like do not depend on enzyme digestion connection operation of a template, and the method has the characteristics of simplicity and low cost and gradually becomes a hot spot of current research. However, three types of non-target molecules are inevitably generated in the experimental process of the existing random priming PCR (I) non-target products mediated by two specific primers, (II) non-target products mediated by the specific primers and the random primers, and (III) non-target products completely mediated by the random primers. These non-target products can affect the purity of the target fragment and thus the accuracy of the walking result. Whereas class I and II non-target products cannot be amplified in secondary and tertiary PCR due to the lack of binding sites for the second and third specific primers, and are diluted in subsequent reactions. Thus, how to eliminate the effects of class III non-target products has become an important point in random priming PCR applications. Disclosure of Invention In order to solve the problems in the background art, the invention provides a genome walking method based on zigzag PCR, a kit and application thereof. The method can effectively remove the influence of class III non-target products through the unique design of the random primer serrated difference, can not only improve the amplification success rate of complex or high background samples, but also remarkably improve the sensitivity, specificity and repeatability of PCR amplification, and effectively overcome the amplification difficulty of long fragments or fragments with high GC content. In the application, a brand new random priming PCR-saw tooth PCR (SawTooth PCR, ST-PCR) is established, and meanwhile, the feasibility of ST-PCR is further verified by walking dnaK, pduC, ALDOA genes. In order to achieve the above object, the present invention provides a genome walking method based on zigzag PCR, comprising the steps of: (1) The primary amplification, namely adding a hot start DNA polymerase, a random primer and a specific primer SP1 into a reaction system containing dNTPs and a PCR buffer solution by using a annealing temperature grading cycle for amplification, wherein the annealing temperature grading cycle comprises three annealing stages, namely a first stage comprises 5 high-stringency cycles, a second stage comprises 1 extremely low-stringency cycle and a third stage comprises 25-35 high-stringency cycles; (2) The secondary amplification is carried out by adding random primer and specific primer SP2 into a reaction system containing dNTPs and PCR buffer based on the primary amplification product and carrying out amplification through annealing temperature grading circulation, wherein the annealing temperature grading circulation consists of two annealing stages, namely a first stage comprises 1 very low-stringency circulation and a second stage comprises 25-35 high-stringency circulation; (3) The third-stage amplification, namely adding a random primer and a specific primer SP3 into a reaction system containing dNTPs and a PCR buffer solution based on a second-stage amplification product, and carrying out amplification through an annealing temperature grading cycle, wherein the annealing temperature grading cycle consists of two annealing stages, namely a first stage comprises 1 very low-stringency cycle, and a second stage comprises 25-35 high-stringency cycles; the SP1, the SP2 and the SP3 are nested primers designed based on genome DNA; the random primer is selected from a random primer group at least comprising 3 pieces of primers with Tm value of 58-6