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CN-121991945-A - Method for generating viscous end product in PCR process

CN121991945ACN 121991945 ACN121991945 ACN 121991945ACN-121991945-A

Abstract

The invention belongs to the field of molecular biology, and particularly provides a PCR amplification method capable of directly generating a viscous end product in a PCR process and application thereof. The invention utilizes the novel double-chain sticky end primer to be matched with the high-fidelity 5'-3' -end-free exonuclease active polymerase, and can generate a DNA product with a sticky end structure in one step in the PCR process, thereby obviously improving the efficiency of DNA connection in molecular cloning or sequencing and library construction.

Inventors

  • DENG JIE
  • WANG XIAOYAN
  • DING YIJIE
  • WANG CHENYI
  • JIANG YU
  • CAI LIYI
  • WANG YAN
  • WANG QINGKAI
  • WEI HUIJUAN

Assignees

  • 石家庄乐仁康健医学检验实验室有限公司

Dates

Publication Date
20260508
Application Date
20260303

Claims (10)

  1. 1. A double-stranded cohesive end primer is characterized by comprising a primer strand (P strand) and a blocking strand (B strand), wherein the P strand and the B strand are complementarily bound through a complementary region sequence, and the 3' -end of the B strand has a protruding cohesive end sequence.
  2. 2. The double-stranded cohesive end primer of claim 1, wherein the P-strand comprises a complementary region sequence and a primer region sequence, wherein the primer region sequence is located at the 3' end of the P-strand and is capable of complementary pairing with a target gene template strand.
  3. 3. The double-stranded cohesive end primer according to any one of claims 1-2, wherein the B strand comprises a complementary region sequence and a cohesive end sequence, preferably the complementary region sequence of the B strand binds complementarily to the complementary region sequence of the P strand, more preferably the complementary binding is a complete complementary binding.
  4. 4. The double-stranded cohesive end primer according to any one of claims 1 to 3, wherein the complementary region sequence has a dissolution temperature Tm value higher than the Tm value of the P-strand primer region sequence, preferably the Tm value is higher than 5 to 15 ℃.
  5. 5. The double-stranded cohesive end primer of any one of claims 1-4, wherein the complementary region sequence comprises, but is not limited to, a tag sequence and barcode; preferably, the length of the complementary region sequence is 5-50 bp; More preferably, the primer region sequence length of the P strand is 5-40 bp; further preferably, the cohesive end sequence length of the B chain is 1-10 bp.
  6. 6. A double-stranded cohesive end primer pair comprising the double-stranded cohesive end primer of any one of claims 1-5.
  7. 7. Use of the double stranded cohesive end primer of any one of claims 1-5 for: 1) Application in PCR amplification; 2) Use in the rapid preparation of cohesive end DNA sequences.
  8. 8. A PCR amplification product, characterized in that the PCR amplification product comprises the double-stranded cohesive end primer according to any one of claims 1-5, preferably the PCR amplification product further comprises a DNA polymerase without 5'-3' exonuclease activity, more preferably the product type is a kit.
  9. 9. A PCR amplification method for generating double-stranded cohesive end is characterized in that the PCR amplification method adopts the double-stranded cohesive end primer as set forth in any one of claims 1-5 to amplify nucleic acid, and preferably, DNA polymerase without 5'-3' end exonuclease activity is adopted to carry out polymerization reaction in the amplification process.
  10. 10. A method for ligating double-stranded DNA, characterized in that the method comprises the step of directly ligating a target DNA with the DNA product obtained by the PCR amplification method according to any one of claims 8 to 9, wherein the direct ligation is not required to further generate a cohesive end at the end of the DNA product.

Description

Method for generating viscous end product in PCR process Technical Field The invention belongs to the field of molecular biology, relates to a novel polymerase chain reaction technology, and particularly provides a method for generating a viscous end product in a PCR process and application thereof. Background In molecular biology, the ligation of DNA fragments is a key step in achieving molecular cloning and DNA sequencing banking, wherein the use of cohesive ends (STICKY ENDS) for DNA fragment ligation is an efficient and common method. The cohesive end is usually formed by cutting DNA by restriction endonuclease, has a single-chain protruding structure, can realize specific connection with the complementary end through base pairing, obviously improves the probability of molecular collision and improves the DNA connection efficiency. In both molecular cloning experiments and sequencing and library building, the target fragments to be subjected to DNA ligation are often further ligated after PCR amplification. In the existing method, no matter sequencing and library establishment or molecular cloning experiment flow, restriction enzyme is generally required to be carried out on a PCR product with a flat tail end after PCR, a sticky tail end is generated by enzyme digestion, and then DNA connection is carried out based on the sticky tail end. The above steps generally require three key steps to achieve nucleic acid amplification and ligation, are cumbersome, time-consuming and costly. In addition, enzyme-based methods are often only suitable for longer DNA fragments, and may miscut genomic fragments in whole genome-based second generation sequencing, resulting in fragment loss. Besides the common enzyme digestion method, the method is commonly used for TA cloning, wherein the end repair of the blunt-end PCR product is required, A is added to the 3' -end to form a short sticky end consisting of a single A, and finally DNA ligation is performed under the action of T4 ligase. Although the method avoids errors caused by enzyme digestion, compared with a long-viscosity tail end, the TA connection binding capacity is lower, so that the experimental duration is longer. The present invention has been made in view of the above-described drawbacks of the conventional methods. Disclosure of Invention The invention designs a PCR method based on double-chain sticky end primers and simultaneously matched with high-fidelity 5'-3' -end exonuclease activity, and a PCR product with a sticky end structure at the end can be generated in one step in the PCR process. The structure can directly connect DNA in the subsequent process, omits the process of generating sticky ends by enzyme digestion or end repair, greatly improves the efficiency of connecting DNA in the process of molecular cloning or sequencing and library construction, and saves the cost. Thus, the present invention may include at least the following objects: The first object of the present invention is to provide a primer sequence of a novel structure; A second object of the present invention is to provide a novel PCR amplification technique; A third object of the present invention is to provide a method for generating a viscous end product during PCR; A fourth object of the present invention is to provide a DNA ligation method. In order to achieve the aim of the invention, the invention adopts the following technical scheme: The present invention provides a double-stranded cohesive end primer comprising a primer strand (P strand) and a blocking strand (B strand), the P strand and the B strand being complementarily bound by a sequence of a complementary region, and the 3 '-end of the B strand having a protruding cohesive end sequence (relative to the 5' -end of the P strand). Further, the P strand comprises a complementary region sequence and a primer region sequence, wherein the primer region sequence is positioned at the 3' -end of the P strand and can be complementarily paired with the target gene template strand. Further, the B strand comprises a complementary region sequence and a cohesive end sequence, preferably, the complementary region sequence of the B strand and the complementary region sequence of the P strand are complementarily bound, and more preferably, the complementary region sequence of the B strand and the cohesive end sequence are complementarily bound. In some aspects, the complementary region sequence has a solubility temperature Tm value that is higher than the Tm value of the P-strand primer region sequence; preferably, the Tm of the complementary region sequence is 5-15℃higher than the Tm of the P-strand primer region sequence. In some aspects, the types of the complementary region sequences include, but are not limited to, tag sequences, barcode sequences, or homology arm sequences required for cloning, and the like; In some preferred aspects, the length of the aforementioned complementary region sequence is from 5 to 50 bp; in some pref