Method for synthesizing nucleotide pools in high-throughput manner by aid of semiconductor chips and assembling double-stranded DNA (deoxyribonucleic acid)

Abstract

The invention relates to a method for synthesizing nucleotide pools in a high-throughput manner by the aid of semiconductor chips and assembling double-stranded DNA (deoxyribonucleic acid). The method includes steps of (1), dividing pre-synthetic double-stranded DNA into a plurality of DNA fragments; (2), designing nucleotide by the aid of the DNA fragments; (3), adding sequences to two ends of a primer which is the nucleotide, synthesizing nucleotide sequences and preparing the nucleotide sequences by the aid of the semiconductor chips; (4), carrying out first-round amplification by the aid of the primer with biotin labels; (5), recombining first-round PCR (polymerase chain reaction) products by the aid of at least one type of recombinant enzymes; (6), purifying PCR mixtures; (7), carrying out second-round PCR, and assembling nucleotide fragments to obtain assembled PCR products; (8), carrying out third-round PCR amplification on the PCR products by the aid of designed head and tail primers to obtain double-stranded DNA fragments. The method has the advantages of low cost and high one-step synthesis throughput.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to the technical field of DNA synthesis, and specifically relates to a high-throughput method for synthesizing nucleotide pools and assembling double-stranded DNA on semiconductor chips. Background technique [0002] Synthetic biology is an emerging interdisciplinary subject in the 21st century. It uses engineering research ideas to design and modify genes, biological components, biological pathways, and even the entire genome. It is used in the fields of biology, medicine, environmental protection, energy, and agriculture. It has broad application prospects. Synthetic biology technology mainly includes: synthesis of double-stranded DNA, synthesis of regulatory elements, operon synthesis, gene synthesis, etc. Among them, gene synthesis is the main component of synthetic biology and the basis and skeleton of synthetic biology. Gene synthesis refers to the technology of artificially ...

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Problems solved by technology

[0007] The method commonly used in the current technology is to synthesize nucleotides on a chip and then assemble double-stranded DNA using this as a raw material. The steps of gene synthesis and error repair are cumbersome, the degree of integration and miniaturization is low, and the cost is high. Therefore, a method is now needed Combining semiconductor chip synthesis technology and DNA large fragment assembly technology together, using electrochemical semiconductor chip and DNA large fragment assembly technology to successfully develop a next-generation DNA synthesis platform

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