Filter carrier and application thereof

A carrier and oligonucleotide technology, applied in the field of genetic engineering, can solve the problem of increasing the proportion of wrong sequences

Active Publication Date: 2016-07-13
ADAGENE SUZHOU LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when a gene or its fragments are spliced ​​by multiple oligonucleotides, the proportion of wrong sequences will increase significantly

Method used

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  • Filter carrier and application thereof
  • Filter carrier and application thereof
  • Filter carrier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Construction of filter vector pFV02

[0067] Unless otherwise noted, all primers in the construction process were synthesized and provided by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0068] 1) Amplify the CAT gene

[0069] The CAT gene was amplified from the pACYC184 plasmid (NEB, #VKNO287) template by overlapping PCR, and the BspEI restriction site was mutated.

[0070] Overlap PCR amplification process

[0071] The first step: with the pACYC184 plasmid as a template, the primer pair is catF1 and catR1 (Suzhou Jinweizhi Biotechnology Co., Ltd.), PCR amplification obtains fragment cat-1 (length 560bp) ( figure 1 , (a)).

[0072] Second step: with pACYC184 plasmid as template, primer pair is catF2 and catR2, PCR amplification obtains fragment cat-2 (length 499bp) ( figure 1 ,(a)).

[0073] The 3rd step: take above-mentioned fragment cat-1 and cat-2 as template, primer pair is catF1 and catR2, PCR amplification obtains full-length cat gene ( figure 1...

Embodiment 2

[0132] Example 2. Quality detection and quality improvement of degenerate oligonucleotides using filter carriers

[0133] General process:

[0134] Step 1: Convert the single-stranded degenerate oligonucleotides into double-stranded DNA fragments by PCR. The required primers vary according to the sequence of the degenerate oligonucleotides. The double-stranded DNA fragments are ligated to the pFV02 filter vector.

[0135] Step 2: transform the ligation product into competent bacterial strain DH5α, spread on LB plates with ampicillin (final concentration 50 μg / ml) and / or chloramphenicol (34 μg / ml).

[0136] Step 3: When performing quality detection of degenerate oligonucleotides, randomly select a certain number (usually 12) of colonies grown on LB+chloramphenicol (note: no ampicillin) plates, and in 37 After overnight incubation at °C, plasmid DNA was extracted for sequencing.

[0137] The fourth step: collect all the colonies grown on the LB plate with ampicillin (final co...

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Abstract

The present invention provides a filter carrier for screening an oligonucleotide having a target length, the filter carrier, from 5 'to 3' direction, includes a promoter, a reporter gene, and at least one enzyme digestion site located between the promoter and the reporter gene, and after the oligonucleotide having the target length is inserted into the enzyme digestion site, frameshift mutation of the reporter gene may not be caused. The present invention also provides a construction method of the filter carrier and application of the filter carrier in improvement of correct rate of chemical synthesis oligonucleotides used in a phage or bacterial gene library construction process.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a filter carrier used for chemically synthesized oligonucleotide quality detection and accuracy improvement. More specifically, the present invention discloses a filter carrier for screening oligonucleotides with a target length. The invention also provides the construction method of the filtering carrier and its application in improving the correct rate of the chemically synthesized oligonucleotide used in the construction process of the phage or bacterial gene library. Background technique [0002] Oligonucleotides synthesized by chemical methods are widely used in life sciences, such as artificial synthesis of genes, gene sequencing, and gene amplification by PCR methods. At present, the chemical synthesis of oligonucleotides is mainly through the phosphoramidite chemistry (Phosphoramidite chemistry), and each synthesis cycle includes 4 chemical reactions: deblocking, ligat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/65C12N15/64C12N15/66C12Q1/68
Inventor 杜方勇罗培志
Owner ADAGENE SUZHOU LTD
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