Enhancement of the transfection efficiency of nucleic acids by u

Details Enhancement of the transfection efficiency of nucleic acids by u Enhancement of the transfection efficiency of nucleic acids by u

1996-10-11

1998-08-11

Ketter, James

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Treating animal or plant material or micro-organism

536 2541, C12N 106, C12N 1510

Patent

active

057926514

DESCRIPTION:

BRIEF SUMMARY
The present invention pertains to the use of isopropanol in aqueous solutions for enhancing the transfection efficiency of nucleic acids in prokaryotic and eukaryotic cells.
Applicant's DE 36 39 949 A1 pertains to a process for the isolation and purification of nucleic acids, in particular long-chain nucleic acids. This involves the use of aqueous solutions (buffers) of relatively low ionic strength to wash the nucleic acid which is first adsorbed at low ionic strength on an anion exchange matrix. Thereafter, the nucleic acids are desorbed from the matrix using buffers with higher ionic strengths. Ethanol, inter alia, may be employed for the precipitation of nucleic acids, especially DNA. Solutions containing lower alcohols are proposed in Applicant's P 43 21 904. The solutions containing lower aliphatic alcohols are used together with high concentrations of chaotropic ions to adsorb nucleic acids on inorganic materials which have not been modified by anion exchanging groups.
Then, the nucleic acid thus isolated is frequently introduced in eukaryotic or prokaryotic cells by chemical or physical methods. This process, called transfection, is used in order to study the regulatory effect of certain genes or their expression products on the genome of the host cell. Such methods involve rather high expenditures of both work and time since a period of 10 to 14 days must be scheduled, as a rule, for performing and evaluating a single experiment. Therefore, a high interest exists in seeing, for example, that the plasmid DNA to be studied is introduced in as many cells of a cell culture as possible. The frequency of intake of foreign DNA offered, which is called transfection efficiency, depends on a variety of factors. Usually, the transfection efficiency depends, in addition to the quality of the cell culture and the transfection method, substantially on the quality or purity of the plasmid DNA used.
In Ehlert et al., Biotechniques 14, 1993, 546, it could be shown that plasmid DNA prepared using the QIAGEN.RTM. anion exchange material according to the method described in DE 36 39 949 will give a transfection efficiency which is improved by 70% over that of DNA purified by cesium chloride density gradient centrifugation.
Surprisingly, the transfection rate known from Biotechniques 14, 1993, 546, could be enhanced by up to 20% by using isopropanol in aqueous solutions which are employed for the isolation of nucleic acids.
Thus, the present invention pertains to a process for the isolation of nucleic acids which has an improved transfection rate over that known from DE 36 39 949 A1 by using aqueous solutions containing isopropanol for the separation and isolation of nucleic acids on anion exchangers.
In a preferred embodiment, the isopropanol will be present in the aqueous solutions in an amount of from 1 to 50% by volume, in particular from 5 to 25% by volume, particularly preferred from 10 to 15% by volume. The aqueous solutions may also include, in particular, salts of monovalent or divalent cations commonly used for anion exchange chromatography, such as sodium chloride, potassium chloride etc., or combinations thereof. Preferably, the aqueous solutions are buffered with buffer materials conventionally used in molecular biology, such as TRIS/HCl, MOPS etc. and have pH values of between 6 and 9.
The use of isopropanol according to the invention can be applied, in particular, in the isolation of DNA, such as plasmid DNA, but is not restricted to such types of DNA.
The nucleic acids obtained using isopropanol according to the invention are particularly suitable for being employed in gene-therapy methods.
According to the invention, solutions are also claimed which may contain isopropanol in amounts of from 5 to 60% by volume. These are, in particular, the washing buffers which can be used according to DE 36 39 949 A1. Those buffers contain 0.5 to 1.5M sodium chloride or potassium chloride and 20 to 80 mM MOPS or TRIS/HCl at a pH value of 6 to 8. According to the invention, aqueous solutions are also claimed includin

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