For example, the genomes of many viruses (hepatitis C, influenza virus, picornaviruses, etc.

For example, the genomes of many viruses (hepatitis C, influenza virus, picornaviruses, etc.

Due to this sequence, which is present in the clinical preparation in a minimal amount (one or more copies) and cannot be detected by any other methods, can be easily detected by PCR. PCR allows you to find only one abnormal sequence per 100,000-1000000 normal cells.

The exponential 40 best narrative essay topics increase in the number of copies of the target molecule not only provides a high sensitivity of the method, but also facilitates their detection. Each round of PCR takes from 2 to 5 minutes, and usually to achieve the required sensitivity is enough 25-50 rounds, ie 2-4 hours. Thus, the entire analysis can be performed in one day. In addition, since the content of PCR products is quite large, you can use non-isotopic detection methods.

In the table. 1 shows data to compare the PCR method with other molecular genetic research methods. It is seen that it has two important advantages: high sensitivity and short analysis.

Table 1. Comparison of different methods of hybridization

 

PCR

Southern blot hybridization

In situ hybridization

Sensitivity

1/100000

1/100

10 targets

Specificity

High

High

High

Duration of analysis

1 day

1 day

1 day

 

RNA amplification

The possibility of using RNA as a target for PCR significantly expands the range of applications of this method. For example, the genomes of many viruses (hepatitis C, influenza virus, picornaviruses, etc.) are represented by RNA. However, in their life cycles there is no intermediate phase of transformation into DNA. To detect RNA, it is first necessary to translate it into the form of DNA.

To do this, use reverse transcriptase, isolated from two different viruses: avian myeloblastosis virus and Moloney murine leukemia virus. The use of these enzymes is associated with some difficulties. First of all, they are thermolabile and therefore can be used at a temperature not exceeding 42 ° C, because at this temperature RNA molecules easily form secondary structures, the reaction efficiency is significantly reduced and according to various estimates is approximately 5%.

Attempts are being made to circumvent this shortcoming by using as inverted transcriptase a thermostable polymerase derived from the thermophilic microorganism Thermus Thermophilus, which exhibits transcriptase activity in the presence of Mn2 +. It is the only known enzyme capable of exhibiting both polymerase and transcriptase activity.

To carry out the reverse transcription reaction in the reaction mixture as well as in PCR must be present primers as a fuse and a mixture of 4 dNTF as a building material.

After the reverse transcription reaction, the resulting cDNA molecules can serve as a target for PCR.

Analysis of PCR-amplified DNA

Various methods are used to analyze PCR-amplified DNA. We will consider the three simplest: gel electrophoresis, dot blot hybridization and Southern blot hybridization. They can be used to analyze most PCR products, but absolutely accurate results can only be obtained by sequencing.

Preparation of PCR products

First of all, it is necessary to remove the mineral oil that covered the reaction mixture. To do this, a drop of chloroform is added to the PCR tube, the tube is shaken and centrifuged at 12,000 g for 1 min to separate the aqueous phase containing the PCR products. Amplified; DNA can be stored with chloroform at 4 ° C for several weeks. For the subsequent analysis usually take from 1/10 to 1/5 of the volume of the reaction mixture.

When using DNA isolation from blood cells, preparatory work is not carried out.

Gel electrophoresis

Agarose gel electrophoresis makes it easy, without the use of radioisotopes, to find amplified DNA and determine its size. Let’s focus on some of its features in relation to the analysis of PCR-amplified DNA. 10-20 μl of amplified DNA is separated in a 2% agarose gel with the addition of a special DNA dye, such as ethidium bromide, together with standard fragments of 50-1000 bp.

When filling with the help of combs, special holes are formed in the gel, into which amplification products are further introduced. When using DNA isolated from blood cells use 5 μl of amplified DNA mixed with 3 μl of dyes on the parafilm and applied to the well.

The gel plate is placed in an apparatus for horizontal gel electrophoresis and a DC voltage source is connected. Negatively charged DNA begins to move in the gel from minus to plus. In this case, shorter DNA molecules move faster than long ones. The rate of DNA movement in the gel is affected by the concentration of agarose, electric field strength, temperature, composition of the electrophoresis buffer and, to a lesser extent, the HC composition of DNA.

All molecules of the same size move at the same speed. The dye is incorporated (intercalated) by plane groups into DNA molecules. After electrophoresis, lasting from 10 minutes up to 1 hour, the gel is placed on a transilluminator filter that emits light in the ultraviolet range (254 – 310 nm). The ultraviolet energy absorbed by the DNA in the region of 260 nm is transferred to the dye, causing it to fluoresce in the orange-red region of the visible spectrum (590 nm).

The brightness of the bands of the amplification products may be different. Therefore, it is often accepted in PCR laboratories to evaluate the result on a three-four or five-point system. However, as noted earlier, this cannot be attributed to the initial amount of target DNA in the sample. Frequent decrease in the brightness of the glow of the bands is associated with a decrease in the efficiency of amplification under the influence of inhibitors or other factors.

Electrophoresis is performed at high voltage (10-15 V / cm), because the small fragments formed by PCR are difficult to detect after electrophoresis at night at low voltage due to their intense diffusion. Separation can be enhanced using NuSieve polyacrylamide or agarose gels (FMC Bio-Products, Rockland, USA) with a high concentration of agarose (3-4%). However, if the analysis needs to be performed quickly and at low cost, 2% agarose gels are quite acceptable.

Usually, when amplification of DNA isolated from fixed tissues, the yield of PCR products is lower and they are less specific than in the case of amplification of highly purified DNA.

Instructions 1 for the preparation of agarose gel (50 ml).

Agarose gel consists of 1.8% agarose and 98.2% Tris buffer.

Weigh the agarose (0, 9g).

Transfer the agarose to a heat-resistant flask.

Add 50 ml of Tris buffer to the flask.

Install the die, install the comb on the die.

Melt the gooseberry by heating in a microwave oven to obtain a homogeneous solution.

Add the dye ethidium bromide to the flask and mix.

Pour the agarose gel on the die.

Hybridization of PCR-amplified Southern DNA

This method allows to identify bands in the gel observed after electrophoresis of amplified DNA. Both isotopically and non-isotopically labeled probes are used for hybridization. The method of hybridization of PCR products is described in detail in protocol 1.

Protocol 1. Hybridization of PCR-amplified Southern DNA

Materials:

Buffer for denaturation: 1.5 M NaOH, 0.5 M NaCl Buffer for neutralization: 1.5 M Tris-Hc1, 0.5 M NaCl, p 6.5 20 x SSPE: 3.6 M NaCl, 0.2 M sodium phosphate, 0.02 M EDTA, pH 7.4 10% (v / o) LTO

Method:

For denaturation of DNA fragments, the gel is soaked in denaturation buffer with gentle shaking for 30 minutes. at room temperature, then kept in buffer for neutralization for 30 minutes During this time, soak a nylon filter to transfer DNA in 6 x SSPE for 5 minutes. (or longer). We use Genetrans 45 filters (Fiasco, Wolburn, Massachusetts, USA). Assemble the DNA transfer system, and carry out the transfer at room temperature overnight. 20 x SSPE is used as a transfer buffer. At the end of the transfer, remove the filter paper and note the position of the holes on the filter. The filter is removed and washed to remove sticky agarose in 20 x SSPE for 1 min. Sew the DNA to the filter, placing the latter (side up with DNA) for 5 minutes. under a UV lamp (254 nm). You can also use a special device for DNA suturing (Stratagene, La Jolla, California, USA). Place the filter in a plastic bag and seal it. Carry out pre-hybridization at 42 0C for 10 minutes and hybridization with a suitable labeled probe at the same temperature for 30-60 minutes 5-polynucleotide kinase-labeled oligonucleotides are commonly used as probes. Wash the filter in two or three shifts of 2 x SSPE, 0.1% LTO, for 5 minutes in each shift. Sometimes at this stage washing is carried out in more severe conditions, but the need for this procedure is determined experimentally. The hybridized probe is detected by radioautography or non-isotopic detection methods.

Dot blot hybridization of PCR-amplified DNA

Dot blot hybridization gives a simple answer of the type yes and no and is especially useful in cases where the analysis of a large number of samples. One example of the application of this method is described in Protocol 2.

Protocol 2. Dot-blot hybridization of PCR-amplified DNA

Materials:

Solution for denaturation: 0.4 M NaOH, 1 mm EDTA. 20 x SSPE (see protocol 15.7). Nylon filter. Device for obtaining dot blots.

Method:

Soak a nylon filter in distilled water for at least 5 minutes. 2.10-20 μl of PCR-amplified DNA is added to 300 μl. solution for denaturation (denaturation occurs almost instantly). Each sample is introduced into a separate cell of the device to obtain dot blots; make sure that it takes about 1 minute to absorb the sample from the cell. Wash each cell 20 x SSPE three times for 5 minutes. Remove the filter from the appliance and rinse it in 20 x SSPE. Perform the operations described in protocol 15.7, starting with paragraph 5.

Interpretation of results

The PCR result can be qualified as positive or negative depending on whether the target of interest to us is found in the sample or not. However, disruption of the normal course of amplification, insufficient sensitivity of the method and unforeseen polymorphism of the target sequence in the binding of primers or hybridization probe can give a false negative result.

In the case of contamination of the samples and random homology between the probe, primers and the sequence similar to the target, false negative results are obtained.

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