CS269549B1 - Spoaob to increase the efficiency of DNA transfer to bacterial cells - Google Patents

Abstract

The solution relates to a method of increasing the efficiency of the transfer of ζΝΑ into bacterial cells by using compounds of general formulae I. and II., where R. = a hydrocarbon chain with a number of carbons from 6 to 20, straight or branched, n = 2 to }2, X » Cl, Br, I., which are added to the reaction mixture when the cells are not in a competent state in an amount of 0.0001 to 0.5 mg/ml. The solution can be used in the field of gene engineering, modern biotechnologies, in the creation of gene banks, and wherever increased transfer of DNA into recipient cells is required.

Description

The invention relates to a method for increasing the efficiency of the transfer of deoxyribonucleic acid (DNA) into a host cell. By using substances of the general formulae

CH_ I ^J

CH ₃ - ⁺ N - CH ₃ ,x“ *1 (I)

CH ₃ ch ₃

CH ₃ - * N - (CH ₂ ) _n - * N - CH ₃ . 2X where Rj · hydrocarbon chain with a number of carbons 6 to 20, straight or branched, optionally containing heteroatomdm, n = 2 to 12, X » Cl, Br, I,

The transfer of hybrid DNA molecules in vitro into new host cells is an essential part of genetic engineering manipulation. The goal of this procedure is to clone foreign genetic information in recipient cells, propagate it, and select it, with the subsequent selection of suitable clones.

Gene transfer into bacterial cells can be accomplished in three basic ways: transformation, or transfection - i.e., direct uptake of free DNA by recipient cells, transduction, in which DNA is transferred by phage from one cell to another, and conjugation, in which DNA is transferred by direct contact between bacterial cells. The result is a heritable change in the recipient cell.

A bacterium that is able to take up exogenous free DNA and incorporate it into its genome is called competent. For some bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Bacillus subtilis) competence is a natural physiological state during the growth cycle, while for many bacteria (e.g., Escherichia coli), yeasts, and also for animal cells cultured in vitro, competence must be artificially induced by physicochemical procedures.

The disadvantage of the current methods is that for the most commonly used host cells (E. coli) it is necessary to induce competence in a relatively complicated way, while the efficiency of transformation (number of transformants per introduced amount of DNA) is low. Similarly, the uptake of large plasmids (> 30 kb) by competent cells is inefficient,

Transformation of E. coli with free DNA is very important for the technology of DNA cloning, because competent cells can be relatively easily transformed with various small replicons, which are used for the construction of recombinant DNA molecules. In addition, the transfer of cloned genes into a genetically new environment is also an important stage of their analysis. Increasing the efficiency of cell transformation with exogenous DNA can be used in the field of genetic engineering and modern biotechnology, when creating gene banks in microorganisms.

CS 269 549 Bl

The essence of the solution according to the invention lies in the use of surface-active cationic substances, which, when added to the reaction mixture, increase the efficiency of the transformation by up to 10 times.

The advantage of the solution according to the invention is that with a slight modification of the usual transformation method, it is possible to obtain a multiple amount of transformants using the same amount of isolated DNA.

The following examples illustrate, but do not limit, the use of the invention.

Example 1 ml of an exponential culture of E.coli DH1 in LB medium is resuspended in 5 ml of a CaCl ₂ solution (0.05 mol/l) after centrifugation (rotation frequency 7000 min/l, 4 °C, 10 min) and left for 20 min. at 0 °C. After further centrifugation and resuspension in 1 ml of a cold CaCl ₂ solution (0.05 mol/l), the suspension is distributed into 200 ml tubes.

To the sample in the test tube, 20/.1 of the stock solution of the detergent NN*-bis (octyldimethyl)-1,6-hexanediammoniumdibromide (final concentration 20 _/ «g/ml) is added, and after 10 min, 4 °C, the DNA solution (plasmid pAT153) is added so that there is 1^g of DNA in the test tube. The mixture is incubated at 0 °C for 30 min, then heated to 42 °C for 2 min, 0.75 ml of LB broth is added and after 30 min at 37 °C, 10//1 of the suspension is plated onto Petri dishes containing nutrient agar with ampicillin. Only cells into which the plasmid pAT153 has been introduced grow. No detergent was added to the control samples. Results see table, 1. Example 2

The sample was processed as in Example 1, but instead of N,N'-bis(octyldimethyl)1,6-hexanediammonium dibromide, the same amount of N,N'-bis(pentadecyldimethyl)-1,6-hexanediammonium dibromide (2 µg/ml) was added. Results see Table 1.

Example 3

The sample was processed as in Example 1, instead of plasmid pAT153, plasmid pBR322 and octyldimethylammonium iodide (0.1 mg/ml) were added to E. coli BH101.

See Table 1. .

Example 4

The sample was processed as in Example 1, but instead of N,N*-bis (octyldimethyl)-1,6-hexanediammonium dibromide, n,Π - bis (te

CS 269 549 Bl ·

(tradecyldimethyl)-1,6-hexanediammonium dibromide (20 µg/ml), and DNA obtained by rapid method (pUC9) was added instead of highly purified DNA (pAT153). The number of cells containing the plasmid was determined by plating on MacConkey agar, on which they formed red colonies.

Results see table 1.

TABLE 1

Bacteria DNA Substance (concentration, mg/ml) Number of transformants per Petri dish with substance control Ss83bsb*=£«3xismi3ΧΒ··αη BBZB3SXKBX8B3XΒΠS3MB3SS3SCR3 E.coli DH1 pAT153 ^C 8 (0.02) 123 28 E.coli OHI PAT153 ^C 15 (0.02) 55 28 E. coli 0M83 pUC9 ^C 8 (0.02) 22 2 E. coli 0M83 pUC9 ^C 14 (0.02) 12 2 E.coli HB1O1 PBR322 ^C VIII (0.1) 10 5 E. coli BH101 PBR322 ^C 8 (0.0005) 14 5 S.typhimurium PBR322 ^C 14 (0.125) 93 0

Cg - N,N'-bis(octyldimethyl)-1,6-hexanediamenium dibromide

C^ ₄ - N,N*-bis(tetradecyldimethyl)-1,6-hexanediammonium dibromide c ₁₅ - nX-bis(pentadecyldimethyl)-1,6-hexanediammonium dibromide Cyjjj - octyldimethylammonium iodide

Claims (1)

TRANSFER OF THE INVENTION · A method for increasing the efficiency of DNA transfer into a bacterial cell, characterized in that substances of the general formula ^CH 3 are used as active ingredients causing an increase in the efficiency of DNA transfer. I CH ₃ - * N - CH ₃ .x ch ₃ «1 • (I) ch ₃ ch ₃ / / _ - * N - (CH ₂ ) _n - * N - CH ₃ ,2X ^R 1 *1 , (II) where R^ is a hydrocarbon chain with a number of carbons from 6 to 20, straight or branched, n has a value of 2 to 12, X means Cl, Br, I, which are added to the reaction mixture when inducing a state of competence in cells in an amount of 0.0001 to 0.5 mg/ml.