SU1538126A1 - Method of detecting nucleic acids - Google Patents

Abstract

The invention relates to molecular biology and genetics and can be used in other areas of biology and medicine for the non-radioactive treatment of nucleic acids. The purpose of the invention is to simplify and reduce the cost of the method of non-radioactive treatment and detection of nucleic acids. The essence of the invention is that, as a label, a hydrazide of the general formula R - CO - NH - NH _{2 is} added to the nucleic acid, where R: HH ₂ -NH-

NH ₂ -NH-CO- (CH ₂ ) _N -

N ₃ - @ - and for the detection of the label, the modified nucleic acid is treated with a glycoprotein enzyme previously oxidized with sodium periiodate, followed by determination of the resulting product of the enzymatic reaction.

Description

The lot is determined by the intensity of staining of the resulting product. Homologous nucleic acids are determined in a similar way after their preliminary hybridization with a labeled nucleic acid enzyme.

Example 1. Introduction of a hydrazide label into a nucleic acid.

A. Inclusion at the C-4 position of cytosine. 10 µg of denatured DNA is dissolved in 50 µl of water and 50 µl of an aqueous solution of adipic acid dihydrazide (concentration of 160 mg / ml) is added, mixed with sodium bisulfite (380 mg / ml) or an aqueous solution of carbazide (360 mg / ml ) with sodium bisulfite (380 mg / / ml), the reaction mixture is incubated for 2 hours at 37 ° C and purified using gel filtration on Sephadex G-25.

B. Turning on with photoreagent. 10 µg of DNA was dissolved in 50 µl of water and 10 µl of a 0.001 M solution of A-azido-2-nitrobenzoic acid hydrazide was added. The mixture is irradiated with visible light for 30 minutes and gel filtered on Sephadex G-25.

PRI mme R 2. DNA containing - hydrazide groups and control unlabeled DNA is applied to a nitrocellulose filter in an amount of 1ng-1pg and 25 ng, respectively. Filters are heated for 2 hours at 80 ° C. Next, incubated in buffer A (0.01 M sodium acetate, pH 4.3, 0.1% Tween-20) for. 20 min and treated with preoxidized or non-oxidized peroxidase for 10 min in buffer A without tween-20 at room temperature. The oxidation of peroxidase is carried out by treatment with sodium periodate (4 mg / ml) for 5 minutes after 5

0

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The luloene filter with DNA samples homologous labeled, applied in an amount of 1-20 pg, and non-homologous DNA (25 pg) is treated with 0 B buffer (0.6 M NaCl, 0.002 mg / ml polyvinyl pyrrolidone, 0.07 M sodium phosphate buffer, pH 7.5, 10 μg / ml RNA, 50% formamide) for 1 hour at 37 ° C. Next, hybridization is carried out in buffer B containing DNA labeled with hydrazide (example IA) (100 ng / ml) at 37 ° C for 16 h. Next, the filter is washed with buffer B (0.07 M sodium phosphate, 0 , 1 M NaCl, 0.5% sodium dodecyl sulfate, pH 7.5) at room temperature 3 times for 10 minutes and buffer B containing NaCl at a concentration of 0.05 M at 48 ° C for 1 hour. Then the filters are kept in Buffer A containing 0.1 M NaCl for 30 minutes and treated with an enzyme as described above .. The detection sensitivity is 20-50 pg with no signal on the non-homologous

0 DNA.

PRI me R 3. Filters with applied DNA containing hydrazide groups (0.01 pg - 1 ng) and unlabelled DNA - 50 ng are kept in a buffer

5 g (0.1 M sodium acetate, pH 5.2,

0.5% tween-20) for I h and treated with pre-oxidized / 5-galactoside for 10 minutes in buffer G without tween-20 at room temperature. Galactosidase oxidation is carried out by treatment with sodium periodate (4 mg / ml) for 5 minutes in 0.1 M sodium phosphate buffer, pH 6.5; the reaction is blocked by the addition of glucose. Next, the enzyme is purified by gel filtration. The filters are washed with buffer D (0.1 M sodium phosphate, pH 7.5) with the addition of 0.05% Tween-20, 2 times for 5 minutes each time and once

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rum D (5 min). A mixture of 5-bromo-4-chloro-3-indolyl-galactoside (0.3 mg / ml) and nitro tetra-eole blue (0.4 mg / ml) in buffer D is added. Incubate for 2-4 hours. Sensitivity for determination of labeled DNA is 0.1, pg with no signal on the control sample (50 ng).

The choice of the enzyme glycoprotein using the method is determined by the characteristics of the tasks being solved and may be associated with such characteristics of the enzymes as cost, stability, size, necessary and available substrates, etc., as well as the necessary and ensured level of sensitivity.

The proposed method allows the molecule to be used as a nucleic acid label less than biotin by about 8 times. Since a hydrazide can be attached to a nucleic acid by the same methods as biotin, this reduces the loading of labeled nucleic acid and, consequently, changes in its properties, and also significantly reduces the cost of methods. For example, the cost of equivalent amounts of reagents for labeling nucleic acids by position C-4 cytosine using a hydrazide as a label is reduced by more than 100 times as compared with biotype labeling (catalog of Sigma, USA, 1987, adipic acid dihydrazide and biotin hydrazide are compared). The effect is also achieved due to the rejection of the need to use the streptavidin-enzyme complex. Thus, the cost of the streptavidin-peroxidase conjugate and peroxidase conjugate with the same activity correlates as 20: 1 (Sigma catalog, USA, 1987). Attaching streptavidin to peroxidase increases the molecular weight of the detector molecule by 2.5 times. Therefore, in the proposed method, the molecule-detector has a smaller size, which provides improved opportunities

Editor N. Tupitsa

Compiled by A. Tehred M. Khodan

its penetration to the labeled nucleic acid. This advantage is most important when using labeled nucleic acids for in situ hybridization.

The sensitivity provided by the proposed method is similar to the sensitivity when using biotin and streptavidin-peroxidase conjugate for detection as a label.

Claims (1)

Thus, the proposed method reduces the cost of non-radioactive labeling and detection of nucleic acids and simplifies it, since it allows refusing the use of intermediary molecules (biotin and avidin) and operations for their use. In addition, it improves the detection capabilities with the help of enzymes in non-radioactive labeling, which is caused by a decrease in the molecular weights and sizes of the label on the nucleic acid and the detector compound. Invention Formula A method for detecting nucleic acids, including introducing a label into a nucleic acid, further processing it with an enzyme and determining the amount of the resulting product of the enzymatic reaction, characterized in that, in order to simplify and reduce the cost of the method, a hydrazide of the general formula is used as a label R-CO-NH-NH2, where R-NH -NH-; NHz-NH-CO- (CH4) n; South and the treatment is carried out with an enzyme, a glycoprotein. Proofreader T.Malets