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Oligonucleotide chain random splicing method

An oligonucleotide and long-chain technology, applied in nucleotide libraries, protein nucleotide libraries, organic compound libraries, etc., can solve the problems of inapplicability and inapplicability of multi-fragment splicing, and achieve large storage capacity and guaranteed sequence The effect of polymorphism

Pending Publication Date: 2022-04-12
PEKING UNIV
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Problems solved by technology

[0006] The second is Pre-mix. Due to the limitations of the OBOC method for synthesizing larger peptide libraries, the amino acid premix method is often used to synthesize mixed peptide libraries.
The main principle of the first three methods is to generate complementary sequences on both sides of the DNA fragments or to connect them through overlapping sequences, which requires additional enzyme cutting sites, so additional interference sequences will be introduced. For the purpose of screening short peptide libraries DNA splicing does not apply
The use of PCR technology for DNA splicing requires the design of a 10-25bp complementary region between each fragment. The length of this complementary region often exceeds the length of the oligonucleotide chain to be spliced, so it cannot be applied to shorter Multi-fragment splicing of oligonucleotide strands

Method used

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  • Oligonucleotide chain random splicing method
  • Oligonucleotide chain random splicing method
  • Oligonucleotide chain random splicing method

Examples

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Embodiment 1

[0102] The splicing of embodiment 1 oligonucleotide chain

[0103] In this embodiment, six 12nt DNA fragments are used as oligonucleotide chains to be assembled, and any four oligonucleotide chains (named A, B, C, D) are randomly assembled. The basic process is: use magnetic beads as For the carrier, first connect Smart3_RC to the oligo-dT of the magnetic beads, then connect A, B, C, and D to the magnetic beads in different order, and finally use AdrecF primers and CDSIII_RC primers to combine the spliced ​​long-chain oligonucleotides acid for amplification. The structure of the assembled long-chain oligonucleotide is: oligonucleotide chain A-the first linker-oligonucleotide chain B-the second linker-oligonucleotide chain C-the third linker-oligonucleotide For the nucleotide chain D, the sequences (5'-3') of the first linker, the second linker and the third linker used are GGTGCA, GCTGCA, and GGAGCA, respectively.

[0104] The specific random stitching method is as follows, ...

experiment example

[0182] The random oligonucleotide chain library constructed in the above-mentioned examples and comparative examples was tested for splicing efficiency, storage capacity and polymorphism. The specific process is as follows:

[0183] 1. Library concentration detection

[0184] The spliced ​​final products obtained by PCR amplification cycles of 20 and 33 cycles in Step 9 in Example 1 and Comparative Example 1 were detected by agarose gel electrophoresis. The result is as image 3 As shown, when there is no linker in Comparative Example 1, there is still no clear product band after 33 cycles of amplification, indicating that the product of successful splicing cannot be obtained without a linker, and the splicing efficiency is 0, so subsequent cloning and sequencing detection are not performed ; while embodiment 1 amplified 20 cycles and 33 cycles of the spliced ​​final product obtained clear bands can be seen, indicating that the content of the final product is about 100ng.

...

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Abstract

The invention relates to the technical field of molecular biology, in particular to an oligonucleotide chain random splicing method. According to the random splicing method provided by the invention, any k oligonucleotide chains in n oligonucleotide chains are randomly spliced into long-chain oligonucleotide by taking a magnetic bead as a carrier and adopting a primer group for randomly splicing the oligonucleotide chains. According to the method, the short-chain oligonucleotides can be randomly spliced into the long-chain oligonucleotides at relatively high efficiency, the randomness of the nucleotides in the short-chain oligonucleotides and the polymorphism of the sequences are better ensured by reducing the length of the synthesized nucleotide random sequence, and then the length of the target oligonucleotide chain is reached through random splicing. The constructed long-chain oligonucleotide library and random peptide library have large storage capacity and high polymorphism, and have good application prospects.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer set for random assembly of oligonucleotide chains and a method for random assembly of oligonucleotide chains. Background technique [0002] Peptide libraries are a collection of a large number of short peptides of specific length and different sequences, which include the arrangement and combination of various (or most of) amino acid sequences in short peptides of this length. At present, screening using random peptide libraries has been widely used in many fields such as protein-protein interaction, drug design and screening. [0003] The more commonly used method for synthesizing random peptide libraries is to design nucleotide palindromic sequences during long-chain DNA synthesis, wherein the first two nucleotides encoding random nucleotide sequences are any nucleotide N(A / T / C / G), the last nucleotide is designed as K (G / T) according to the degeneracy of th...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12P19/34C40B50/06C40B40/08
Inventor 夏朋延王硕朱芳蕊钱言
Owner PEKING UNIV
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