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

A nucleotide pool and semiconductor technology, applied in the field of molecular biology, can solve the problems of cumbersome steps, high cost, low degree of integration and miniaturization, etc., and achieve the effect of high throughput and low cost

Inactive Publication Date: 2016-01-27
SUZHOU HONGXUN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 i

Method used

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

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Experimental program
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Embodiment 1

[0039] like figure 1 As shown, a kind of semiconductor chip high-throughput synthetic nucleotide pool and a method for assembling double-stranded DNA are disclosed in Embodiment 1. The method steps are:

[0040] (1) Segment the pre-synthesized double-stranded DNA into multiple 200-1000bp DNA fragments.

[0041] (2) Design nucleotides with the DNA fragment in (1), the length of each nucleotide is 30-90nt.

[0042](3) Add 20nt flank sequences to both ends of the 30-90nt nucleotides designed in (2), use TypeII or TypeIIs restriction endonuclease to recognize and digest the sequence, and synthesize a total length of 80-200nt Nucleotide sequence, wherein the nucleotides are prepared by a semiconductor chip; in this embodiment, the above-mentioned TypeII or TypeIIs restriction endonuclease is used for the high-throughput synthesis of nucleotide pools on the semiconductor chip, and the TypeII restriction endonuclease Enzymes include, but are not limited to: EcoRI, BamhI, HindIII; t...

Embodiment 2

[0049] Example 2 discloses an example of actually synthesizing double-stranded DNA by the method in Example 1.

[0050] The sequence of pre-synthesized DNA being divided into DNA fragments (414bp) in embodiment 2 is as follows:

[0051] GGATCCAGAGCAGAGGAGCCAGCTGTGGGCACCAGTGGCCTCATCTTCCGAGAAGACTTGGACTGGCCTCCAGGCAGCCCACAAGAGCCTCTGTGCCTGGTGGCACTGGGCGGGGACAGCAATGGCAGCAGCTCCCCCCTGCGGGTGGTGGGGGCTCTAAGCGCCTATGAGCAGGCCTTCCTGGGGGCCGTGCAGAGGGCCCGCTGGGGCCCCCGAGACCTGGCCACCTTCGGGGTCTGCAACACCGGTGACAGGCAGGCTGCCTTGCCCTCTCTACGGCGGCTGGGGGCCTGGCTGCGGGACCCTGGGGGGCAGCGCCTGGTGGTCCTACACCTGGAGGAAGTGACCTGGGAGCCAACACCCTCGCTGAGGTTCCAGGAGCCCCCGCCTGGAGGAGCTGGCCCCCCACTCGAG

[0052] Step 1: Primers designed according to the DNA sequence are shown in Table 1:

[0053] Table 1 Design primer list

[0054]

[0055]

[0056] Step 2: Add a 20nt flank sequence and an enzyme recognition site sequence to both ends of the above-mentioned designed primers. In this embodiment, the TypeIIs restriction endonucleas...

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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 ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 刁文一齐金才柳伟强孙德斌杨平
Owner SUZHOU HONGXUN BIOTECH CO LTD
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