Biochemistry laboratory work (Практикум по биохимии). Методическое пособие

1. To remove RNA impurities from the DNA preparation, add 2 µl of 1 mg/ml RNase A to the nucleic acid solution, mix thoroughly by pipetting, and incubate at 37 °C for 1 h.

2. For DNA resuspension, add 1/10 volume (15 µl) of 3M sodium acetate, pH 5.0, mix thoroughly and add 4 volumes (600 µl) of 96% ethanol to the nucleic acid solution and incubate at -20°C for at least 12 h.

3. Precipitate the DNA by centrifugation at 12,000 rpm for 15 min at 4 °C. Carefully remove the supernatant by leaning the end of the tube against a filter paper, and dry the DNA precipitate at room temperature (approximately 30 min). Dissolve the DNA precipitate in 20 µl distilled water.

4. To determine the concentration of extracted DNA, add 2 µl of the DNA solution to 100 µl of water, mix thoroughly, and estimate the optical density at 260 nm. Assess the purity of the extracted DNA by the optical density ratio A260/A280. Store the DNA solution at -20°C.

Laboratory work №4: Determination of FGB/F13A1 levels by TaqMan PCR

Polymerase chain reaction (PCR) is a widely used molecular biology technique that enables the amplification of specific DNA sequences. It has revolutionized various fields, including medical diagnostics, genetic research, forensic analysis, and biotechnology. One of the most significant advancements in PCR technology is the development of real-time PCR, also known as quantitative PCR (qPCR).

Real-time PCR allows for the monitoring and quantification of DNA amplification in real-time, unlike traditional PCR methods that require post-amplification analysis. This technique utilizes fluorescent probes, such as TaqMan probes, to measure the amplification of the target DNA during the PCR reaction. TaqMan

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real-time PCR has emerged as a powerful tool for accurate and sensitive quantification of nucleic acids. The TaqMan probe is a dual-labeled oligonucleotide consisting of a fluorescent reporter dye and a quencher molecule. During the PCR reaction, the TaqMan probe specifically hybridizes to the target DNA sequence, which is amplified by the DNA polymerase enzyme. As the DNA amplification progresses, the polymerase cleaves the TaqMan probe, separating the fluorescent reporter dye from the quencher molecule. This separation results in an increase in fluorescence signal, which is detected and measured in real-time by a fluorescence detection system. The real-time PCR data generated by the TaqMan system allows for the quantification of the initial amount of target DNA present in the sample. By comparing the amplification curves of the target DNA with known standards or control samples, the absolute or relative quantification of the target sequence can be determined. This quantitative information is valuable in various applications, such as gene expression analysis, pathogen detection, and genetic profiling. TaqMan real-time PCR offers several advantages over conventional PCR methods. It provides greater sensitivity, specificity, and accuracy, allowing for the detection of low copy number targets. The real-time monitoring of amplification enables faster and more efficient data acquisition, eliminating the need for post-amplification analysis. Additionally, TaqMan probes can be multiplexed, allowing the simultaneous detection and quantification of multiple target sequences within a single reaction.

The objective of the work is to determine DNA concentration in the sample by TaqMan q-PCR.

Blood clotting is a multistage enzymatic process that involves proteases, non-enzymatic proteins, with fibrin as the final substrate of the process.

Plasma clotting factors are divided into 3 groups:

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•proteins with proteolytic enzyme properties (VII, X, XII, XI, II, XIII);

•proteins without enzymatic properties, enhancers of blood coagulation processes (VIII, V);

•fibrin — final product of the clotting process.

Fibrinogen (I) is a glycoprotein with a molecular weight of about 340 kDa; one of the main parameters characterizing blood coagulation ability. The "assembly" of fibrin goes through several stages: formation of fibrin monomers, polymerization, stabilization of the clot. The fibrin that forms as a result of these processes and forms the basis of the clot is insoluble fibrin. Fibrinogen is produced by the liver, thence it enters the blood.

FGB — gene, encoding the fibrinogen, SNP in it could be a risk factor for peripheral and coronary thrombosis and is associated with the degree of atherosclerotic vascular damage. The risk of thrombosis increases significantly in the presence of triggering factors such as surgery, taking hormonal contraceptives, various diseases (diabetes, cancer, obesity), as well as pregnancy and childbirth.

F13A1 gene encodes subunit A of clotting factor XIII. Factor XIII consists of 2 catalytic A-subunits and 2 B-subunits. Factor XIII is a plasmatic glycoprotein, usually circulating in blood in complex with fibrinogen. Upon cleavage of the activation peptide by thrombin and in the presence of calcium ion, the plasma factor factor XIIIa acts as a transglutaminase to catalyze the formation of glutamyl-lysine crosslinking between fibrin molecules, thus stabilizing the fibrin clot. It also crosslinks α2-plasmin inhibitor, or fibronectin, to the α-chains of fibrin. Mutations in F13A1 mostly decreases the risk of pathologies, linked with increased thrombosis, but it also can cause delayed bleedings.

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What is the study used for?

To evaluate the risk of:

•the complications of many cardiovascular diseases,

•arterial and venous thrombosis,

•the cause of myocardial infarction,

•coronary artery disease,

•pulmonary embolism,

•deep vein thrombosis,

•strokes.

When the study is necessary?

•If you have relatives with thrombotic complications under the age of 50 (deep vein thrombosis, pulmonary embolism, stroke, myocardial infarction, sudden death).

•For pregnancy planning.

•When planning operations (preferably).

•For the selection of hormonal contraceptives.

•In case of increased levels of antiphospholipid antibodies and/or increased levels of homocysteine.

Equipment and materials:

1)UV lamp;

2)Biofuge pico Heraeus centrifuge;

3)iCycler 5 real-time detection amplifier (BIO-RAD, USA);

4)automatic pipettes of variable volume for 1-10, 10-100, and 100-1000 µl;

5)tips for pipettes;

6)0.5 ml. disposable tubes;

7)0.2 ml disposable thin-walled striped tubes;

8)rack for 0.2 ml sterile tubes;

9)stand for 0.5 ml tubes;

10)gloves;

11)ice;

12)cotton swabs.

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Reagents:

1)taq polymerase solution at a concentration of 0.5 u/µl;

2)prepared reaction mixture for TaqMan PCR, containing dNTP, forward and reverse primers, Taq polymerase (Vector Best JSC production);

3)normal homozygote control (manufactured by Vector Best JSC);

4)"mutant homozygote" control (produced by Vector Best JSC);

5)positive control — genomic DNA, at a starting concentration of 50 ng/µl;

6)distilled water;

7)70% ethanol.

Working scheme

Attention! Wear disposable gloves.

1. Preparing to work

1.Wipe the workplace and dispensers with a cotton swab moistened with 70% ethanol and treat with UV light for 20 min.

2.Thaw the PCR buffer at room temperature. Thaw primer solutions and samples at room temperature and store them protected from light (e.g. wrapped in foil).

3.Turn on the iCycler 5 amplifier by pressing the button on the back of the instrument.

2. Course of operation

1. It is recommended to use human genomic DNA as a calibration standard. Prepare and label five 0.5 ml tubes with concentrations of 5 ng/µl, 500, 50, 5, and 0.5 pg/µl. Add 45 µl of water to each test tube.

Prepare serial dilutions of the genomic DNA. Mix the genomic DNA by pipetting and centrifuge for 30 sec at maximum speed.

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Transfer 5 µl of DNA to a tube labeled 5 ng/µl, mix by pipetting half of the volume. Then take 5 µl from this tube and add to the tube labeled 500 pg/µl, mix. Similarly prepare 50, 5 and 0.5 pg/µl calibration standards.

2.Prepare MasterMix in 1.5 ml tubes with the number of test samples (2) + calibration standards (5) and negative control (water) + spare tube (30 µl per reaction × the number of reactions) in a dark place. All measurements must be performed in 4 replicates so that in the further analysis questionable values can be excluded.

3.Add 5 µl of analyzed DNA or calibration DNA or negative control (water) and then 25 µl of "MasterMix" into the 0.2 mL striped tubes.

Note: q-PCR tubes can only be signed on the wall of the tube. The lid must not contain any colored markings.

4.Mix the resulting mixture thoroughly by pipetting, changing the pipette tip for each tube, and centrifuge for 10 sec at maximum speed.

5.Place tubes into the sockets of the amplifier: the strip with the calibration samples should be placed strictly in row "1" in orientation - the tube numbers correspond to the row number in ascending order (tube number 1 - row 1, cells A-B, tube number 2 - row 1, cells C-D, etc.) (Figure 2).

6.Make sure that the walls of the tubes are tightly seated against the walls of the amplifier nests, close the lid of the amplifier.

7.To perform PCR on an iCycler 5 (Bio-Rad, USA) with subsequent data analysis using iQ5 software, select the "FGB/F13A1" program by running the file FGB/F13A1.run. This file specifies the following PCR conditions:

4.5 min — 95 °C — 1 cycle,

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15 sec — 95 °C,

15 sec — 60 °C.

Figure 2. The location of the sample tubes is noted in the tab <PLATE>

8.In the <PLATE> tab, specify the number, location and identifiers of the samples, including the negative control sample, mark the location of the tubes on the thermoblock matrix according to their setting.

9.Click the <SELECT/ADD FLUOROFORE> button on the right side of the window, select in addition to the FAM fluorophore / the TAMRA fluorophore. For the new fluorophore, repeat in the <PLATE> tab the number, location and sample identifiers, including the negative control sample, mark the position of the tubes on the thermoblock matrix according to their setting.

10.Start the PCR by successively pressing the <RUN> and <BEGIN RUN> buttons. In the suggested window, select the directory for saving the file with the result.

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3. Registration of amplification results

1.The instrument records the fluorescence signal automatically during amplification.

2.Detection and recording of results is performed by the amplifier detecting in accordance with the instrument's instructions.

3.After the end of the amplification program, an appropriate information message appears on the screen and you will be prompted to proceed to the analysis of the results.

4.The analysis is done automatically. The <RESULTS> tab will indicate the FGB/F13A1 DNA concentrations in µl of the sample according to the number assigned when setting up the reaction.

5.You can generate and print a report based on the results of the analysis.

6.As a result of PCR performed by the instrument a calibration curve will be drawn, which will be used to calculate the concentration of DNA in the test sample (figure 5).

4. Accounting for the results of a reaction

1.Reaction results are presented as concentrations (number of pg/µl) of DNA. Results that lie within the calibration concentration range are considered valid.

2.For biological samples that do not contain the DNA of the sequence being detected and the negative control sample, the detection amplifier registers a concentration below 2 pg or no signal.

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3.

Figure 5. Calibration curve representing cycle threshold vs. matrix concentration

The reaction result is considered invalid and samples should be reanalyzed if:

-the amplification efficiency reflected under the <E> calibration plot lies outside the 90-110% range;

-the regression coefficient for the calibration curve <R^2> is outside the range 0.985-1.000;

-the concentration of the negative control sample exceeds the threshold level of 2.5 pg.

4. An unreliable result may be due to the presence of inhibitors in the DNA preparation obtained from clinical material; incorrect implementation of the analysis protocol; non-compliance with the amplification temperature regime, etc. In this case a repeat PCR with the given DNA preparation is required, or a repeated isolation of DNA or a repeated collection of clinical material (performed sequentially).

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