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Method for detecting DNA (deoxyribonucleic acid) content of CHO (cholesterol) cells by probe

A cell and probe technology, applied in the field of quantitative detection of CHO cell DNA content, can solve the problems of high implementation cost and low sensitivity, and achieve the effect of high specificity and good sensitivity

Active Publication Date: 2011-08-17
SHANGHAI HENLIUS BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The fluorescent PCR method of the present invention adopts Taqman probe technology, overcomes the shortcomings of traditional detection methods such as low sensitivity and high implementation cost, and provides a simple, cheap, and high-accuracy quantitative fluorescent PCR detection method and a kit for the method

Method used

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  • Method for detecting DNA (deoxyribonucleic acid) content of CHO (cholesterol) cells by probe
  • Method for detecting DNA (deoxyribonucleic acid) content of CHO (cholesterol) cells by probe
  • Method for detecting DNA (deoxyribonucleic acid) content of CHO (cholesterol) cells by probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Design of primers and preparation of reference materials for the detection of DNA residues in CHO cells by fluorescent quantitative PCR

[0037] 1. Materials and reagents

[0038] DNTPs, Taq DNA polymerase, fluorescent dyes and PCR buffer are commercially available products. The 7500Fast fluorescent quantitative PCR instrument is a product of Applied Biosystems in the United States. The DNA diluent is prepared by our laboratory using analytical reagents. Product of Bio-Tek Corporation.

[0039] 2. Design and synthesis of primers and probes

[0040] Using the Genebank accession sequence (Genebank accession number EF540878.1) as a template, use Primer Primer 5.0 software (Premier Biosoft, USA) to design PCR primers and probes, and select the following combinations from the preliminary experiments.

[0041] The nucleotide sequence of Primer-F is: 5'-ACAGGTTTCTGCTTCTGGCT-3';

[0042] The nucleotide sequence of Primer-R is: 5'-TAGCAGACACTGTTGTAGAG-3';

[0043] Probe: 5'-...

Embodiment 2

[0052] Example 2 Application of Fluorescent Quantitative PCR to Detect CHO Cell DNA Concentration in Analysis of CHO Cell Gene Copy Number

[0053] 1. The purpose is to provide an internal reference for analyzing the gene copy number of the target gene.

[0054] 2. Preparation of CHO cell DNA

[0055] Collect CHO cells of different cell lines and different passage times, use genomic DNA extraction kit to extract genomic DNA, first measure the concentration of extracted genomic DNA by spectrophotometry, and dilute the samples appropriately so that each sample is within the range of the standard curve .

[0056] 3. Select 7 concentrations of reference substances, the concentrations of which are 3.0×106fg / μl, 3.0×105fg / μl, 3.0×104fg / μl, 3.0×103fg / μl, 3.0×102fg / μl, 3.0×101fg / μl , 3.0fg / μl Refer to the method described in Example 1 for quantitative PCR analysis. Amplification results and standard curves such as image 3 , Figure 4 shown.

[0057] 4. The expression of the tar...

Embodiment 3

[0058] Example 3 Application and Specificity Analysis of Fluorescent Quantitative PCR Detection of Residual DNA in CHO Cells

[0059] 1. Preparation includes the following PCR amplification reaction solution and dilution solution:

[0060] PCR system: PCR buffer (10X) 3.0μl; dNTP (2mM / L) 2.0μl; MgCl 2 (50mM / L) 2.0μl; Rox dye (50X) 0.6μl; forward primer (10μmol / L) 1.0μl; reverse primer (10μmol / L) 1.0μl; probe (10μmol / L) 1.5μl Taq enzyme ( 50U / μl) 0.1 μl; template 9.5 μl; pure water 9.3 μl.

[0061] DNA diluent: use deionized water as solvent, Tris-HCl concentration is 5.0mM / L, EDTA concentration is 1.0mM / L, adjust pH to 8.0 with NaOH solution.

[0062] 2. Sample collection, transportation and storage

[0063] Sample collection: including semi-finished and finished products of recombinant protein drugs produced by using CHO cells as host cells, which are operated according to the sampling requirements of the National Institute for the Control of Biological Products, sealed an...

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Abstract

The invention discloses a method for detecting DNA (deoxyribonucleic acid) content of CHO (cholesterol) cells by a probe, which comprises the following steps of: adopting a nucleotide sequence in a gene bank with accession number of EF 540878 so as to design a specific primer and a Taqman probe, carrying out the real-time fluorescence quantitative PCR (polymerase chain reaction) after extracting the DNA (deoxyribonucleic acid) from a sample under test, and obtaining the DNA (deoxyribonucleic acid) content of the CHO (cholesterol) cells in the sample under test by calculation according to an obtained standard curve. The invention further discloses a kit based on the method. The invention can accurately and quantificationally detect the residual DNA (deoxyribonucleic acid) content of CHO (cholesterol) cells in products derived from the CHO (cholesterol) cells such as therapeutic protein drugs, recombinant vaccines, monoclonal antibodies and the like. The method also provides an internalreference for analyzing copy numbers of specific genes in the CHO (cholesterol) cells.

Description

technical field [0001] The invention relates to a method for quantitatively detecting the content of CHO cell DNA (that is, Chinese hamster ovary cell genome DNA). Background technique [0002] Gene expression systems used in the field of genetic engineering are broadly classified into prokaryotic, yeast, plant, insect and mammalian cell expression systems. Compared with other systems, the advantage of mammalian cell expression system is that it can guide the correct folding of proteins and provide complex glycosylation modifications, so the expression products are closest to natural higher organisms in terms of molecular structure, physical and chemical properties and biological functions. protein molecule. The large-scale production technology of mammalian cells is also becoming more and more mature and perfect. Therefore, the preparation of therapeutic recombinant protein drugs through mammalian cell culture expression has become the mainstream technology in the field o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 郎国竣郭新军姜伟东刘世高周双宬马辰张二辉
Owner SHANGHAI HENLIUS BIOTECH INC
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