SU1555652A1 - Method of determining microquantities of dna - Google Patents

Abstract

The invention relates to biochemistry and molecular biology and can be used to monitor DNA content under conditions of low concentrations of polynucleotide, including its isolation and purification, in radiobiology in assessing the damaging effects of ionizing radiation, as well as in medicine during diagnosis some types of diseases. The aim of the invention is to increase the sensitivity of the method for determining DNA microscopic quantities while cheapening it. The basis of the new method is the use of new fluorescent dye 2- [2- (4-hydroxyphenyl) -5 (6) -benzimidazolyl] -5 (6) - (1-piperazinyl) benzimidazole under conditions optimal for the formation of a specific complex of this dye cDNA Measurement of the fluorescence intensity (IF) is carried out before the dye interacts with the analyzed DNA, after it and after addition of a known concentration of the DNA solution to the same cuvette. The calculation of the desired polynucleotide concentration is carried out according to the formula C (X) = C _Art ^. (N ₁ : N ₂ ) ^. [(IF ₁ -IF ₀ ) :( IF ₂ -IF ₁ )], where C _st is the initial concentration of the standard DNA solution, µg / ml

N ₁ - dilution of the test sample in a cuvette

N ₂ - dilution of a standard DNA solution in a cuvette, IF ₀ - intensity of background fluorescence

IF ₁ - the fluorescence intensity of the colored sample

IF ₂ - fluorescence intensity of the sample after the addition of standard p-ra DNA. The proposed method allows one to determine the DNA content in solutions with a concentration 1.5 to 2 times lower than the known methods using the commercial fluorescent dyes DAFI and Hoechst 33258. Due to the relatively high specificity of the new method, it can be successfully applied when analyzing low concentrations of DNA in biological materials, in particular in blood plasma. 5 tab.

Description

This invention relates to the field of biochemistry and molecular biology and can be used to control

DNA content under conditions of low polynucleotide concentrations, in particular, in radiobiology in assessing the damaging effects of radiation, as well as in medicine in the diagnosis of certain types of diseases.

The purpose of the invention is to increase the sensitivity and reduce the cost of the method for determining DNA microscopic quantities.

The basis of the proposed method is the use of the new fluorescent dye 2 -G2- (4-hydroxy-phenyl) -5 (6) -benzimidazolyl 5 (6) - (1-piperazinyl) -benzimidazole of the formula

Have

 13C H VOH

n n

(I)

under conditions optimal for the formation of a specific complex of this dye with DNA (at pH & 1 + 0.6).

The fluorescent dye (1) is characterized by L6015 350 + 2 nm and & f4vop 460 + 2 nm. Measurement of the fluorescence intensity (IF) is carried out before (IF) and after (IF) the dye interacts (I; with the DNA solution being analyzed, and after adding a known concentration of DNA solution (IF2) to the cuvette. Calculation of the desired concentration, µg / ml, DNA is carried out according to the formula

WITH -

st

HZ p,

IF, - IF0 IF2 - IF,

where C

st

P

P

2

iFo

IF "0

five

0

concentration of standard DNA, µg / ml; diluting the test sample in a measurement cuvette; diluting standard DNA in a measuring cuvette; background fluorescence intensity;

IF, fluorescence intensity, colored (T); the fluorescence intensity of the sample after the addition of a solution of standard DNA. The proposed method allows determining the DNA content in solutions with a concentration of up to 3-10 µg / ml (lower sensitivity limit), which is about 1.5-2 times lower than the sensitivity limit for the known fluorescent dyes DAFI and Hoechst-33258. By virtue of the rather high specificity of the dye (I), the method for determining the DNA microscopic amounts can be used in analyzing the DNA content in biological materials, in particular in blood plasma.

Example 1. Synthesis of fluorescent dye (I) is carried out according to the scheme

Г№Ь / f -ГНыГ; ш тг

HN

/

V

sh, co

3 cho

.MNg PSNzSS

N02 GS-i / NicK

sn

ms

NHo C17HN:

+ rc oo

 -mng jr:

ixW. nt

- in sn3osngi mi

-N

h v

 N

n

rVv JL

lUi-yi- Ac - NQNi)

The purity of the products is controlled by TLC on plates Silufol-UV-254

about

.t-NTVt v ™

 nt

rVvoH ChmW JL ROH uo / c- |;

in the systems: A - chloroform - methanol (9: 1), B - chloroform - methanol - 25%

fresh ammonia (1 6: 4: 1) j B - acetone - hex (1: 2); G - chloroform - acetic acid - methanol (6: 3: 1).

Yield (I) 93%. M.p. 300 ° C.

Found%: M4 410.

CMHMN60.

Calculated,%: F + 410.49.

UV spectrum, , nm (lgЈ) B ethanol: 263 (4.49), 342 ().

Example 2. Determination of DNA content in a sample with an unknown concentration by the proposed and known methods.

In a standard cuvette per 10 mm fluorimeter (the total volume of the cuvette is 4, 0 mm) containing 1.0 ml of 0.01 M tris buffer, 0.01 M of trilon B and 0.1 M NaCl (pH 8.1 added 0.05 ml of the dye solution (I) to a final concentration of rv µg / ml are measured and the background (IF) fluorescence (3fco ,, 6 350 nm;

register 460 nm), then 0.02 ml of DNA solution from rat thymus of unknown concentration is added and the fluorescence intensity is measured again (IF (), then a known amount of the same DNA is added and the fluorescence intensity measurement procedure (IF2) is repeated. After the additions have been made, the contents of the cuvette are mixed for 8-12 seconds. The unknown concentration of DNA in the analyzed sample is calculated according to the indicated formula.

In the embodiment, instead of dye (I), a DAFI (up & rv, ttp 450 nm) fluorescent dye is used, and the reaction is carried out at pH 7.0.

The results of the analysis are shown in table 1.

From the above data it follows that the use of (T) gives reliable results that are close to those obtained by a known method (the differences are no more than 10%).

Example 3. Determination of IF and LIF for dilute DNA solutions.

In a cuvette containing buffer (Example 2) and a dye ((I), DAFI or Hoechst), in a final concentration of 19.6 HO µg / ml, after measuring the IFO, a certain amount of DNA solution from rat thymus with a known concentration is added and after Sivania measure IF ,.

The dependence of 4IF on the concentration of DNA in a sample stained with various dyes is given in Table 2.

0

five

five

YU

five

0

e

0

five

0

The obtained data allow us to conclude that in case of using (I) at all concentrations of DNA greater than 0.3210 emcg / ml, the LIF value is directly proportional to the concentration of DNA in the analyzed solution. When the DNA concentration is below this value, the linearity of the mutual change of the named quantities is disturbed and the measurement error of the LIF PFpr parameter increases. As a result, the DNA concentration of 0.31 O 5 µg / ml can be considered the lower limit of the sensitivity of the method of determination using (I).

In addition, the determination of trace amounts of DNA using dye (T) is more effective than the known ones.

At DNA concentrations below 0.67 x NO mcg / ml IF and, accordingly, DIF when using DAPI have very low values, and the use of Hoechst is almost impossible.

Example 4. Determination of the relative quantum yield for (I) DAFI and Hoechst 33258.

The determination is carried out in a buffer solution of the same composition as in Example 1 (pH for DAFI and Hoechst 7.2, for (I) 8.1). The final concentration of the dye in all cases is 0.09 µg / ml, h} b - 350 nm; slit} p 5 nm; scaling register 15 nm, for DAFI register is 450 nm, and for Khekhste 33258 and for (I) register 460 nm.

The results of the analysis are given in table 3.

As follows from table 3, the quantum yield of the proposed dye (I) is 1.46 + 0.12 times higher than the known dyes Hochst-33258 and DAFI.

Example 5. Determination of the dependence of the DIF on pH.

The composition of the buffer solution as in Example 1. The concentration of the dye (I) was 1 µg / ml. The concentration of DNA in the sample is 9.4 µg / ml.

The data obtained are given in table 4.

From Table 4 it follows that DIF has a maximum and a constant value at a pH of from 7.5 to 8.7.

Example 6: Determination of DNA content in biological material.

Blood plasma of white run-of-breed mice 22 hours after gamma irradiation at a dose of 8 g and blood plasma intct

animals with a volume of 0.02-0.05 ml. any pretreatment is added to a standard cuvette per 10 mm fluorometer measurement containing 1.0 ml buffer (0.01 M tris, 0.01 W trilon B, 0 , 1 M sodium chloride pH 8.1) and determine the intensity of the fluorescence of the IF0 at a 350nm gap B} B 5 nm. registration - 460 nm slit register nm. After that, 0.05 ml of dye (I) is added, the initial concentration is 2 µg / ml and the IF is measured (|, then 0.02 ml of rat thymus DNA solution with a known concentration (3.7 µg /) is added to the same cuvette. ml) and determine IFG.

The results of measuring the experiments are given in table.5.

Measuring the DNA concentration in the plasma of mice by the proposed method allows one to reveal the known phenomenon of a decrease in the DNA level after irradiation.

The minimum amount of DNA determined in these cases in the cuvette is 0.372 µg / ml for plasma of irradiated mice, and 0.066 µg / ml for reference DNA.

Note. The dilution is carried out at n 53.5 and n2 54.5.

table 2

DAFI

Hechest (I)

0

five

five

The use of the invention will provide an opportunity to carry out a quick, simple and economical analysis of the DNA content in solutions with concentrations 1.5-2 times lower than when using the most sensitive fluorescent dyes known.

Claims (1)

Invention Formula A method for determining micro DNA amounts, including treating a polydeoxyribonucleotide solution with a fluorescent dye, determining the fluorescence intensity of the resulting complex and comparing it with the fluorescence intensity of a standard DNA solution, characterized in that, 5 (6) -benzimidazolylTp5 (6) - (1 -pylerasinyl) -benzimidazol, and the reaction with DNA is carried out at pH 7.5-8.7. Table 1 Table 3 Table 4