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CN-121992069-A - Method for constructing high-throughput library of synthetic genes and verifying sequencing

CN121992069ACN 121992069 ACN121992069 ACN 121992069ACN-121992069-A

Abstract

The application provides an amplification library-building method for high-throughput sequencing verification of synthetic genes. The amplification library construction method comprises the steps of obtaining a joint connection product of which two ends are connected with double-chain joints after a synthetic gene sequence to be detected, carrying out first PCR amplification by using the joint connection product as a template and adopting a first gene specific primer to obtain a first amplification product, carrying out second PCR amplification by using the first amplification product as a template and adopting a second gene specific primer to obtain a second amplification product, carrying out heat denaturation single-chain separation and single-chain cyclization on the second amplification product to obtain single-chain annular DNA, carrying out DNB preparation on the single-chain annular DNA to obtain DNB nanospheres, and sequencing the DNB nanospheres to obtain synthetic gene sequence information. The method verifies that the flux of the synthesized genes is high, and can meet the flux requirement of large-batch gene synthesis.

Inventors

  • HUANG FEI
  • CHEN TAI
  • WANG YUN
  • SUN PEI
  • SHEN YUE
  • ZHANG WENWEI
  • Jiang Xianger

Assignees

  • 常州新一产生命科技有限公司
  • 华大工程生物学长荡湖研究所
  • 深圳华大生命科学研究院

Dates

Publication Date
20260508
Application Date
20241105

Claims (9)

  1. 1. The amplification and library-building method for high-throughput sequencing verification of synthetic genes is characterized by comprising the following steps of: obtaining a joint connection product of which two ends are connected with double-chain joints after a synthetic gene sequence to be detected, wherein the joint connection product comprises an optionally existing target area and a non-target area; Performing a first PCR amplification using the adaptor-ligated product as a template and a first gene-specific primer that binds to the target region to obtain a first amplified product, and Performing second PCR amplification by using the first amplification product as a template and adopting a second gene specific primer to obtain a second amplification product, wherein the second gene specific primer is combined with the target region; performing heat denaturation single-strand separation and single-strand cyclization on the second amplification product to obtain single-strand circular DNA; preparing the single-stranded annular DNA by DNB to obtain DNB nanospheres; and sequencing the DNB nanospheres to obtain the synthetic gene sequence information.
  2. 2. The method of claim 1, wherein the target region comprises a plurality of target genes, the first gene-specific primer comprises a plurality of primers that bind to the plurality of target genes, respectively, and the second gene-specific primer comprises a plurality of primers that are inner nested primers of the first gene-specific primer that bind to the plurality of target genes, respectively.
  3. 3. The method of claim 1, wherein the second gene-specific primer comprises a moiety that binds to the target region and a moiety that is at the 5' end and that is identical or partially identical to the second strand of the double-stranded adaptor.
  4. 4. The method according to claim 1, wherein the number of cycles of the first PCR amplification is 10 to 30 cycles, preferably 20 cycles, and the number of cycles of the second PCR amplification is 30 to 40 cycles, preferably 35 cycles.
  5. 5. The method of claim 1, wherein the target region comprises one or more of a vector homology arm base sequence, an index tagged base sequence, and a single stranded circularization primer matched base sequence.
  6. 6. The method of claim 1, wherein the first PCR amplification is one or more of forward amplification and reverse amplification.
  7. 7. The method of claim 1, wherein the first gene-specific primer and the second gene-specific primer are set forth in SEQ ID NOS.1-6.
  8. 8. The method of claim 7, wherein the first gene-specific primer is set forth in SEQ ID NOS.1-4.
  9. 9. The method of claim 7, wherein the second gene-specific primer is set forth in SEQ ID NO. 5-6.

Description

Method for constructing high-throughput library of synthetic genes and verifying sequencing Technical Field The application belongs to the technical field of biology, in particular to the field of high-throughput sequencing of synthetic genes, and more particularly relates to a method for constructing a high-throughput library of synthetic genes and verifying sequencing. Background With the development of genetic science and genetic engineering, DNA synthesis technology plays an increasingly important role in the field of life science. De novo synthesis of DNA elements, including regulatory sequences, whole genes, artificial metabolic pathways, and even complete artificial genomes, will bring about tremendous revolution in human life science research. Since 1961 the first synthesis of oligonucleotide chains by humans (Nirenberg et al (1961) Proc. Natl Acad. Sci. USA 54:1588), DNA synthesis and assembly techniques have been developed. In 2004, tian Jingdong et al succeeded in synthesizing 292 different oligonucleotide strands on a DNA chip and assembling them into a 14.6kb DNA fragment using an oligonucleotide microarray chip, making possible efficient and low cost large-scale synthesis and assembly of long DNA fragments based on chip technology (Tian (2004) Nature 432:1050). In 2010 Kosuri et al realized the synthesis of hundreds of genes using a commercial DNA microarray chip of agilent (Kosuri et al. (2010) nat. Biotech. 28:1295) and provided a complete and complete technical route (Eroshenko et al. (2012) curr. Protoc. In chem. Biol. 4:1). Based on this related art, in 2012, GEN9 is established in the united states as a business company that provides DNA chip synthesis services worldwide for the first time, and the selling price of DNA synthesis products is about $0.26 per bp, which is lower than the market price of conventional DNA synthesis. Two major challenges facing DNA chip synthesis technology are the high error rate of oligonucleotide chains and the impact of the high complexity of oligonucleotide libraries on assembly, respectively. How to further improve the synthesis accuracy of the DNA chip synthesis technology, reduce the assembly and screening cost, and play a vital role in commercial application. In the existing technical route of DNA chip synthesis technology, the correctness detection and correct fragment screening of DNA synthesis fragments are often completed by Sanger sequencing, which is the first generation sequencing technology. Because of the technical bottleneck of DNA chip synthesis, the assembled product often has complex composition, and compared with the designed sequence, each assembled DNA molecule can have multiple variations (including single base variation, nucleotide insertion, deletion and the like) with higher complexity in the sequence, and the probability of single base variation is about 1 per mill to 1 percent due to platform and design difference. Therefore, when the single base error rate is 5 per mill, a 750bp DNA fragment is assembled, the probability of obtaining a completely correct DNA fragment at one time is only 2.33%, the probability of generating more than one internal error is 11.11%, and the probability of generating more than two internal errors is 27.63%. To select the correct molecules with the actual sequence completely consistent with the designed sequence in the complex DNA assembly product, and ensure a success rate of 90%, 98 monoclonal are selected to carry out sanger sequencing, and when errors exceed three, the cost of debugging materials and time is high, and the cost advantage of the DNA chip synthesis technology is lost. Therefore, the traditional method for detecting the correctness of the DNA chip synthesis product based on Sanger sequencing has higher cost, is a main cost source of the DNA chip synthesis technology, and is a significant bottleneck for further reducing the cost of the technology. . Disclosure of Invention The invention provides an amplification library construction method for high-throughput sequencing verification of synthetic genes, which successfully applies a high-throughput sequencing technology to detection of DNA chip synthetic products through a library construction strategy, and greatly reduces the cost of product detection and correct product screening, thereby greatly reducing the cost of DNA chip synthesis. The invention is realized by the following technical scheme: A high throughput detection method of DNA synthesis products, comprising the steps of: obtaining a joint connection product of which two ends are connected with double-chain joints after a synthetic gene sequence to be detected, wherein the joint connection product comprises an optionally existing target area and a non-target area; Performing a first PCR amplification using the adaptor-ligated product as a template and a first gene-specific primer that binds to the target region to obtain a first amplified product, and And carrying out second P