MD707Z - Method for identifying the polymorphic sequences 4a/4b of the nitric oxide endothelial synthetase gene - Google Patents

Abstract

The invention relates to the medical molecular genetics, namely to a method for identifying the polymorphic sequences 4a/4b of the nitric oxide endothelial synthetase gene.Summary of the method consists in that is carried out the whole blood sampling, is extracted DNA with 0.7 M ammonium hydroxide, is maintained at room temperature for 5 minutes and incubated in a thermostat at the temperature of 100°C for 30 minutes with continuous stirring, then the extracted the DNA is amplified using sense NOS3: 5'-GCCCTATGGTAGTAGTGCCTTT-3' and antisense NOS3 primers: 5'-SCTCTTAGTGCTGTGGTCA-3', at the same time is carried out the initial denaturation at the temperature of 94°C for 3 min, then are performed 30 cycles with denaturation at the temperature of 94°C for 1 min, at the temperature of 54°C for 30 seconds, at the temperature of 72°C for 30 seconds and at the temperature of 72°C for 5 minutes, are separated the amplified DNA fragments in 1.8% agarose gel, are stained with 0.5 µg/mL solution of ethidium bromide and are identified the polymorphic sequences.

Description

The invention relates to molecular medical genetics, namely to a method for identifying polymorphic sequences 4a/4b of the endothelial nitric oxide synthase gene.

Under physiological conditions, nitric oxide (NO) acts as a mediator of several biological functions, one of the most important being the adaptation of the vascular system to the increased metabolic needs after physical exertion. Excess NO under pathological conditions induces peripheral vasodilation and syncope, and nitric oxide deficiency has been associated with such pathologies as hypertension, atherosclerosis and ischemic heart disease. It has been demonstrated that the concentration of NO in the blood depends on the activity of the endothelial nitric oxide synthase gene (eNOS), the latter depending on the genetic polymorphism that encodes it.

The development of harmless, yet effective, procedures for genetic diagnosis and prognosis of coronary diseases by identifying the endothelial nitric oxide synthase (eNOS) gene polymorphism is one of the current problems in modern clinical medicine.

A procedure for genetic diagnosis of polymorphisms in intron 4 of the gene encoding endothelial NO synthase is known, by polymerase chain reaction, where DNA is extracted from the peripheral blood of patients with cardiovascular diseases. In intron 4 of the eNOS gene there is the 4a/4b polymorphism, which contains 4 or 5 repeats of 27 bp (base pairs). This substitution facilitates the potential for enzyme cleavage at this site, diminishing the functionality of the eNOS gene.

To amplify the DNA portion containing the polymorphic nucleotide sequence eNOS-4a/4b, the sense (s) and antisense (as) primers with the following sequences were used:

1) NOS3 (s) 5 ́-AGGCCCTATGGTAGTAGTGCCTTTT -3 ́ ;

2) NOS3 (as) 5 ́-TCTCTTAGTGCTGTGGTCAT-3 ́.

Amplification of the DNA fragment, containing the polymorphic minisatellite marker eNOS-4a/4b, was performed in 50 µL solution: Master Mix (Fermentas equivalent) (2×) - 25 µL, sense primer - 1 pM/µL - 5 µL, antisense primer - 1 pM/µL - 5 µL, genomic DNA - 5 ng/µL - 10 µL, ultrapure H2O - 5 µL.

The composition of the Master Mix solution is as follows: solution for performing PCR (polymerization chain reaction) containing MgCl2 - in a concentration of 2.0 mM - 4.0 µL; deoxynucleoside triphosphate-5 ́-triphosphate (dNTP) in a concentration of 0.4 mM - 0.4 µL; Taq polymerase in a concentration of 0.05 units/µL - 2.0 µL, ultrapure H2O - 18.6 µL.

PCR was performed using the Crocodil-III thermocycler (Appligene S.A., France) using the automatic amplification program with the following regime:

first cycle - 94°C/3 min, next 35 cycles - 94°C/1 min, 60°C/1 min, 72°C/1 min, last cycle - 72°C/6 min. Allele identification was performed after separation of amplified DNA fragments in 2% agarose gel and staining with ethidium bromide solution (0.5 µg/mL).

The amplicon of allele 4a was 393 nucleotide pairs (pn) long, and that of allele 4b was 420 pn [1].

The disadvantages of the described process are that it is expensive, and also:

1) involves DNA isolation using expensive reagent kits; 2) the recommended primers form a large number of dimers during the amplification process; 3) the length of NOS3 primers is large (25 bp); PCR is performed in a volume of 50 µL of the mixture, thus using a large amount of chemical reagents, as well as DNA isolated from the blood of patients. The same program of the PCR method in the thermocycler requires a long period of time - 114 min. For visualization of PCR products, 2% agarose gel is used, which also presents an excessive consumption of reagents, which can be dangerous to the health of investigators in the absence of special protective equipment and techniques. All this requires considerable expenses and contributes to the delay of analyzes and the timely establishment of clinical diagnosis.

The problem solved by the present invention consists in increasing the efficiency of the method for identifying polymorphic sequences 4a/4b of the endothelial nitric oxide synthase gene, as well as in making it cheaper and faster.

The essence of the method consists in collecting a whole blood sample, extracting DNA with 0.7 M ammonium hydroxide, keeping it at room temperature for 5 min and incubating it in a thermostat at 100ºC for 30 min with continuous shaking, then the extracted DNA is amplified using the primers NOS3 sense: 5 ́-GCCCTATGGTAGTAGTGCCTTT-3 ́ and NOS3 antisense: 5 ́- CCTCTTAGTGCTGTGGTCA-3 ́, at the same time the initial denaturation is performed at a temperature of 94°C for 3 min, then 30 cycles are performed with denaturation at a temperature of 94°C for 1 min, at a temperature of 54°C for 30 s, at a temperature of 72°C for 30 s and at a temperature of 72°C for 5 min, the amplified DNA fragments are separated in an agarose gel of 1.8%, stained with a 0.5 µg/mL ethidium bromide solution and identified the polymorphic sequences.

The modification of the primers was designed to reduce the cost and increase the efficiency of amplification of DNA fragments. The modeling for the modification of the primers was performed using the Oligo Analyzer 3.1 software. At all stages of the modeling, the classical laws known for the design of primers were respected.

The modification of the NOS3 (s) primer was achieved in one step by deleting the AG nucleotide from the 5' end and the first T nucleotide from the 3' end:

NOS3 (s) 5 ́-AGGCCCTATGGTAGTAGTGCCTTTT -3 ́ NOS3_modif_s: 5 ́-GCCCTATGGTAGTAGTGCCTT-3 ́

The modification of the NOS3 (as) primer was performed in 2 steps. In order to improve melting, the T nucleotide at the 5' end was replaced with the C nucleotide. In the second step, the first T nucleotide at the 3' end was eliminated in order to shorten the length and reduce the cost of the primer.

NOS3 (as) 5 ́-TCTCTTAGTGCTGTGGTCAT-3 ́ NOS3_modif_as: 5 ́- CCTCTTAGTGCTGTGGTCA-3 ́

The result of the invention consists in making the given method cheaper by using lower concentrations of the reaction components, as well as in accelerating it by reducing the reaction cycles in the PCR thermocycler, which makes it more efficient.

Examples of embodiments of the invention

Example 1

To perform the proposed method, peripheral blood leukocytes collected from 10 patients with ischemic heart disease were used. A total of 10 tests were performed in 3 repetitions to analyze the efficiency of the proposed method in comparison with the closest solution.

Venous blood sampling and leukocyte separation from peripheral blood were performed using the standard method.

The proposed method was performed as follows: DNA was extracted from whole blood leukocytes released by boiling with ammonium hydroxide (MD 3539).

DNA separation program: 10 µL of solution containing about 10,000 leukocytes were mixed with 100 µL of 0.7 М ammonium hydroxide solution in Eppendorf tubes and kept at room temperature for 5 min. Then the closed tubes were incubated in a thermostat at 100°C with continuous shaking for 30 min. The concentration of isolated DNA was checked using a spectrophotometer.

Extracted DNA was amplified by PCR using the primers NOS3 sense (s): 5'-GCCCTATGGTAGTAGTGCCTTT-3' and NOS3 antisense (as): 5'- CCTCTTAGTGCTGTGGTCA-3'.

To carry out the reaction, reagents from Fermentas Inc. were used. 2 experiments were carried out in parallel, tested according to the proposed method and the closest solution.

DNA amplification was performed in a solution volume of 25 µL.

The PCR amplification mixture contains, µL:

Master Mix solution - 12.5,

sense primer with a concentration of 0.75 pM/µL - 1.25,

antisense primer with a concentration of 0.75 pM/µL - 1.25,

Genomic DNA with a concentration of 5 ng/µL - 5,

Ultrapure H2O - 5 µL;

at the same time, the composition of the Master Mix solution contains, µL:

MgCl2 with a concentration of 2.0 mM - 2.0,

dNTP with a concentration of 0.4 mM- 0.2,

Taq polymerase with a concentration of 0.05 Units/µl - 1.0,

Ultrapure H2O - 9.3.

The PCR reaction was performed using a thermal cycler using the automatic amplification program with the following regime:

first cycle - 94°C/3 min, next 30 cycles - 94°C/1 min, 54°C/30 s, 72°C/30 s, last cycle - 72°C/5 min. Allele identification was performed after separation of amplified DNA fragments in 1.8% agarose gel which is stained with an ethidium bromide solution (0.5 µg/mL).

In all 10 patients, the amplicon of allele 4a was 393 bp long, and that of allele 4b was 420 bp long.

In parallel, research was conducted for comparison studies in accordance with the closest solution.

The efficiency of the method for identifying polymorphic sequences 4a/4b of the endothelial nitric oxide synthase gene was evaluated by comparing the number (%) of positive PCR products with the closest solution (tab. 1).

Comparative characteristic of the method according to the invention

and the method according to the closest solution

Table 1

Method, invention Method, closest solution PCR Master mix Final volume 25 µL Final volume 50 µL • Master Mix11 - 12.5 µL • sense primer NOS3_modif_s (in concentration of 0.75 pM/µL) - 1.25 µL • antisense primer NOS3_modif_as (in concentration of 0.75 pM / µL) - 1.25 µL • ultrapure H2O - 5 µL • genomic DNA (in concentration of 5 ng/ µL) - 5 µL • Master Mix12 - 25 µL • sense primer NOS3 (s) (in concentration of 1.0 pM/µL) - 5.0 µL • antisense primer NOS3 (as) (in concentration of 1 pM/µL) - 5.0 µL • ultrapure H2O - 5 µL • genomic DNA (in concentration of 5 ng/ µL) - 10 µL Amplification regime • first cycle - 94°C/3 min • first cycle - 94°C/3 min • next 30 cycles - 94°C/1 min, 54°C/30 s, 72°C/30 s • next 35 cycles - 94°C/1 min, 60°C/1 min, 72°C/1 min • last cycle - 72°C/5 min • last cycle - 72°C/6 min Visualization of PCR products in agarose gel • allele identification was performed after separation of amplified DNA fragments in 1.8% agarose gel, which were stained with an ethidium bromide solution (0.5 µg/mL) • allele identification was performed after separation of amplified DNA fragments in 2% agarose gel, which were stained with an ethidium bromide solution (0.5 µg/mL) The amplicon of allele 4a was 393 bp long, and that of allele 4b was 420 bp long.

When assessing the reaction results, positive amplification was scored 1, and negative amplification was scored 0. The data in Table 2 demonstrate that the experimental results are absolutely identical, therefore, the requested method is not only not inferior to the prototype method in efficiency, but is superior in terms of financial and time savings.

Table 2

Comparative analysis of the results of amplification of the gene encoding endothelial synthetase

of nitric oxide

No. of amplification Success rate of the PCR reaction, performed by the proposed method Success rate of the PCR reaction, performed by the closest method Presence of PCR product Absence of PCR product Presence of PCR product Absence of PCR product I 1 0 1 0 II 1 0 1 0 III 1 0 1 0 IV 1 0 1 0 V 1 0 1 0 VI 1 0 1 0 VII 0 1 0 1 VIII 1 0 1 0 IX 1 0 1 0 X 1 0 1 0 Total 9 1 9 1

Example 2

To test the improved primers against the known primers according to the closest solution, the Oligo Analyzer 3.1 software and the international genetic database GenBank were used.

A. Comparison of sense primers: NOS3 (s) 5'-TCTCTTAGTGCTGTGGTCAT-3' and NOS3_modif_s 5'-GCCCTATGGTAGTAGTGCCTTT-3'.

The comparison of the molecular-biological parameters of the primers was performed using genetic data from the international GenBank database through the BLAST analysis method.

The results demonstrate 100% identity of both primers with human genomic DNA and 77% coverage (primers homologous to the DNA sequence deposited in the database under the number AL110203). Thus, both primers can be used in the PCR reaction as sense primers.

The comparison of the physical parameters of both primers is presented in Table 3.

Table 3

NOS3 parameters/primers (s) NOS3_modif_s Primer length, bp 25 22 Melting temperature 59.6ºC 56.4ºC Size of thermodynamic coefficient of stable secondary structure -1.37ºC -0.22ºC Amount of dimers 13 12

Comparison of primer parameters reveals shorter length of primer NOS3_modif_s compared to NOS3 (s). Thus, synthesis of improved primer with shorter length by 3 nucleotides by the proposed method allows financial savings. A lower melting point compared to the original saves energy costs required in the reaction. The improved primer has a smaller number of dimers, as well as unstable secondary structures, reducing the number of non-specific amplifications during PCR.

B. Comparison of antisense primers: NOS3 (as) 5 ́-TCTCTTAGTGCTGTGGTCAT-3 ́ and NOS3_modif_as: 5 ́- CCTCTTAGTGCTGTGGTCA-3 ́

The results demonstrate 100% identity of both primers NOS3 (as) and NOS3_modif_as with human genomic DNA, encoding intron 4 of the NOS3 gene, and coverage of 95...100% (primers homologous to the DNA sequence deposited in the database under the number NOS3). Thus, both primers can be used in the PCR reaction as antisense primers. In addition, the improved primer has 5% higher affinity for intron 4 of the NOS3 gene.

The comparison of the physical parameters of both primers is presented in Table 4.

Table 4

Parameters/primers NOS3 (as) NOS3_modif_as Primer length, bp 20 19 Melting temperature 53.1ºC 54.0ºC Size of thermodynamic coefficient of stable secondary structure +1.44ºC +0.8ºC Amount of dimers 9 9

Comparison of primer parameters reveals the larger size of the NOS3_modif_as primer compared to NOS3 (as). Thus, the synthesis of the improved primer with a shorter length by one nucleotide allows for financial savings. The improved primer has more unstable secondary structures, which reduces the number of non-specific amplifications in PCR.

C. PCR modeling using modified primer pairs NOS3_modif_s / NOS3_modif_as and known primers NOS3 (s) / NOS3 (as).

According to the known rules for PCR, the difference between the melting temperatures of the sense-antisense primers must be minimal. Thus, the modified primers best satisfy this requirement. The modified primer pair requires a lower melting temperature, saving energy costs for heating the amplifier (Table 5).

Considering that the size of the assumed amplification products will be 420 bp, in accordance with the general amplification rules, the melting and elongation time can be shortened, which will reduce the reaction time.

Table 5

Parameters/primers NOS3 (s) / NOS3 (as) NOS3_modif_s / NOS3_modif_as Difference between primer melting temperatures 6.5ºC (59.6ºC - 53.1ºC) 2.4ºC (56.4ºC - 54.0ºC) Primer melting temperature 60°C 54°C Modeling of the PCR amplification program first cycle - 94°C/3 min, next 35 cycles - 94°C/1 min, 60°C/1 min, 72°C/1 min, last cycle - 72°C / 6 min. first cycle - 94°C/3 min, next 30 cycles - 94°C/1 min, 54°C/30 s, 72°C/30 s, last cycle - 72°C/5 min.

Thus, the method for identifying polymorphic sequences 4a/4b of the endothelial nitric oxide synthase gene is more efficient, cheaper and of shorter duration.

1. Caproş N., Istrati V., Popescu V., Butovscaia C., Popovici I. Polymorphism of the endothelial nitric oxide synthase gene in patients with acute coronary syndrome. Scientific Annals of the State University of Medicine and Pharmacy "N.Testemiţanu". Current issues in internal medicine. University Days 21-23 October, 10th ed., Chişinău, 2009, vol. III, p. 57-62

Claims (1)

Method for identifying polymorphic sequences 4a/4b of the endothelial nitric oxide synthase gene, which consists in collecting a whole blood sample, extracting DNA with 0.7 M ammonium hydroxide, maintaining it at room temperature for 5 min and incubating it in a thermostat at 100ºC for 30 min with continuous shaking, then the extracted DNA is amplified using the primers NOS3 sense: 5 ́-GCCCTATGGTAGTAGTGCCTTT-3 ́ and NOS3 antisense: 5 ́- CCTCTTAGTGCTGTGGTCA-3 ́ in a mixture containing, in µL: Master Mix solution 12.5 sense primer in a concentration of 0.75 pM/µL 1.25 antisense primer in a concentration of 0.75 pM/µL 1.25 genomic DNA in a concentration of 5 ng/µL 5 ultrapure H2O 5, also the Master Mix solution contains, in µL: MgCl2 at a concentration of 2.0 mM 2.0 dNTP at a concentration of 0.4 mM 0.2 Taq polymerase at a concentration of 0.05 units/µL 1.0 ultrapure H2O 9.3, at the same time, initial denaturation is performed at a temperature of 94°C for 3 min, then 30 cycles are performed with denaturation at a temperature of 94°C for 1 min, at a temperature of 54°C for 30 s, at a temperature of 72°C for 30 s and at a temperature of 72°C for 5 min, the amplified DNA fragments are separated in a 1.8% agarose gel, stained with a 0.5 µg/mL ethidium bromide solution and the polymorphic sequences are identified.