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Method for purifying plasmid DNA

A plasmid and drug technology, applied in the field of nucleic acid purification, can solve the problems of insufficient separation, time-consuming and cost-consuming, and difficulties.

Inactive Publication Date: 2011-04-27
AVENTIS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently known methods do not adequately and cost-effectively separate supercoiled and gapped (or relaxed) DNA
Moreover, many known methods have a disadvantage: they use PEG or other additives, which are undesirable in plasmid DNA production because they require additional isolation, handling and quality control methods, which can be difficult and costly. more time and cost
[0022] However, there is a problem in using conventional methods that nucleic acids, especially plasmids, cannot be obtained in a sufficiently high purity and in a sufficiently large amount

Method used

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  • Method for purifying plasmid DNA
  • Method for purifying plasmid DNA
  • Method for purifying plasmid DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0216] The diameter was adjusted for the flow rate used based on the calculation of the Reynolds number (Reynolds) in the coils of the continuous lysis system. Since the following analysis assumes Newtonian behavior of the fluid, the numbers reported below are fully valid only in Bla and to some extent in B2.

[0217] From the value of the Reynolds number, one skilled in the art can determine the type of behavior encountered. Here, we are only concerned with fluids flowing in pipes (hydraulic engineering).

[0218] 1) Non-Newtonian fluid

[0219] The two most commonly encountered non-Newtonian fluids in industry are Bingham fluids and Ostwald de Waele fluids.

[0220] Here, the Reynolds number (Re) is calculated as follows:

[0221] Re N is the generalized Reynolds number

[0222] Re N =(1 / (2 n-3 ))x(n / 3n+1) n x((ρx D n w 2-n ) / m) (1)

[0223] D: Inner diameter of cross section (m)

[0224] ρ: volumetric mass of the fluid (kg / m 3 )

[0225] w: spatial velocity o...

Embodiment 2

[0270] We can split the CL system into 5 steps. In a specific implementation, the configuration is as follows:

[0271] 1) Mixing: cells (in solution 1) + solution 2 (M1 + 3m of 6mm tube). SDS lyses cells to begin, as long as the DNA is not denatured and there is no risk of fragmentation.

[0272] 2) After the lysis is completed, the gDNA is denatured (13m 16mm tube).

[0273] 3) Mixing: lysate + solution 3 (6 mm tube of M2 + 3m).

[0274] 4) Collect the neutralized lysate at 4°C.

[0275] 5) Settling flocs and large fragments of gDNA overnight at 4°C.

[0276] Continuous lysis can be performed using the following conditions:

[0277] - Solution 1: EDTA 10 mM, Glucose (Glc) 9 g / l and Tris HCl 25 mM, pH 7.2.

[0278] - Solution 2: SDS 1% and NaOH 0.2N.

[0279] - Solution 3: acetic acid 2M and potassium acetate 3M.

[0280] - Flow rate 60 l / h: solution 1 and solution 2.

[0281] - Flow rate 90 l / h: solution 3.

[0282] - The cells are adjusted to 38.5 g / l with solutio...

Embodiment 3

[0300] The column used was a 1 ml HiTrap column activated with NHS (N-hydroxysuccinimide, Pharmacia), connected to a peristaltic pump (output 2 The sequence of the group is as follows: 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3' (SEQ ID NO: 1).

[0301] The buffers used in this example are as follows:

[0302] Coupling buffer: 0.2M NaHCO 3 , 0.5M NaCl, pH 8.3.

[0303] Buffer A: 0.5M ethanolamine, 0.5M NaCl, pH 8.3.

[0304] Buffer B: 0.1M Acetate, 0.5M NaCl, pH 4.

[0305] The column was washed with 6 ml of 1 mM HCl, then the oligonucleotide (50 nmol in 1 ml) diluted in coupling buffer was applied to the column for 30 minutes at room temperature. Wash 3 times successively with 6 ml of buffer A and 6 ml of buffer B. The oligonucleotide is thus covalently bound to the column via a CONH bond. Store the column at 4 °C in PBS, 0.1% NaN 3 Medium, and can be used at least 4 times.

[0306] The following two oligonucleotides were synthesized: oligonucleotide 4817:

[0307] 5'-GATCCGAAGAA...

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Abstract

This invention provides a process for the continuous alkaline lysis of a bacterial suspension in order to harvest pDNA. It further provides for optional additional purification steps, including lysate filtration, anion exchange chromatography, triplex affinity chromatography, and hydrophobic interaction chromatography. These optional purification steps can be combined with the continuous lysis in order to produce a highly purified pDNA product substantially free of gDNA, RNA, protein, endotoxin, and other contaminants.

Description

[0001] This application is a divisional application, the filing date of the original application is April 19, 2005, the application number is 200580011784.7 (PCT / EP2005 / 005213), and the title of the invention is "method for purifying plasmid DNA". technical field [0002] The present invention relates to methods for the purification of nucleic acids. In particular, the present invention relates to methods for preparing highly purified plasmid DNA (pDNA), particularly to the production and isolation of pharmaceutical grade plasmid DNA. Background technique [0003] Advances in molecular biology clearly illustrate that plasmid-based therapies, especially in the fields of vaccines and human gene therapy, may support effective disease treatment avenues. However, a significant obstacle to this technique is the preparation of plasmid DNA of sufficient quantity and quality for clinical use. Plasmid DNA is a promising approach to safely and efficiently introduce normal genes into h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N1/06C12N15/10
CPCC02F2303/06A61K48/0091C12P19/34C12N15/101A61P35/00C12N1/10
Inventor 弗朗西斯·布兰切迈克尔·库代尔尼古拉斯·梅斯特拉里蒂瑞·吉尔明戴维·盖拉克
Owner AVENTIS PHARMA INC
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