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

in 1971. However, at that time the main feature of PCR had not yet been demonstrated – an exponential increase in the number of copies of the original DNA fragment as a result of the reaction.

This was done in 1985 by Cetus. Subsequent use of thermostable DNA polymerase in PCR has significantly expanded the possibilities of its application, both for scientific purposes and in the clinic.

In 1985, Saiki et al. published an article describing the amplification of the genomic sequence of b-globin. From that moment on, the number of publications in which the authors reported the use of PCR in their work began to increase exponentially. The method has become so popular that today it is difficult to imagine work in the field of molecular biology without its use.

The method of polymerase chain reaction has developed especially rapidly thanks to the international program "Human Genome". Modern laser sequencing technologies (decoding of nucleotide DNA sequences) have been created. If in the recent past it took a week to decipher a 250-nucleotide DNA sequence (250 bp), modern laser sequencers can detect up to 5,000 bp. a day. This in turn contributes to the significant growth of information databases containing DNA sequences.

Currently, various modifications of PCR are proposed, the possibility of creating test systems for the detection of microorganisms, the detection of point mutations, described dozens of different applications of the method. We will briefly consider the theoretical foundations of PCR and the application of this method for molecular genetic study of tissue samples. The focus will be on practical aspects important for the analysis of conventional tissue samples.

Polymerase chain reaction

PCR is an in vitro specific nucleic acid amplification initiated by synthetic oligonucleotide primers; its main stages are presented in fig. 2. PCR cycle consists of thermal denaturation of DNA, its annealing with a primer and chain elongation (elongation); the change of these stages occurs as a result of a simple change in temperature.

The primers are oriented on the matrix so that the number of rounds of replication grows exponentially, and the number of copies of a specific nucleotide sequence increases accordingly.

The use of molecular methods for clinical diagnosis is limited by their low sensitivity and duration of analysis. Thus, to detect a target nucleic acid by in situ hybridization using a radiolabeled probe, it is necessary that the target be present in the drug in several thousand copies. Frequent number of abnormal sequences in the clinical preparation is diagnostically significant, but less than this value and hybridization can give a false negative result.

In contrast, PCR can detect a unique nucleotide sequence. To do this, in the reaction mixture is added in large excess specific for this sequence oligonucleotide primers ("amplimers"), forming a complex with it, and carry out DNA replication in vitro. Because the amplimers hybridize to both strands of DNA, both the native sequence and the synthesized PCR products can serve as matrices in subsequent rounds of replication, resulting in an exponentially increasing number of copies of the unique sequence.

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 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% https://123helpme.me/write-my-lab-report/ 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