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Basics of PCR. Principles of PCR diagnostics

Recently, a reliable, highly sensitive and rapid method for diagnosing various human infectious diseases has been developed. This method is called "PCR analysis". What is it, what is its essence, what microorganisms it can reveal and how to take it correctly, we will tell in our article.

Discovery history

Also, PCR methods are used in the diagnosis of cancer.

Advantages of the method

PCR diagnostics has several advantages:

High sensitivity. Even in the presence of only a few molecules of microorganism DNA, PCR analysis determines the presence of infection. The method will help with chronic and latently occurring diseases. Often in such cases, the microorganism is otherwise uncultured.
Any material is suitable for research, for example, saliva, blood, genital secretions, hair, epithelial cells. The most common is a blood test and a urogenital smear for PCR.
Long-term cultivation of crops is not required. The automated diagnostic process allows you to get the results of the study after 4-5 hours.
The method is almost 100% reliable. Only isolated cases of false-negative results have been recorded.
Possibility to identify several types of pathogens from one material sample. This not only speeds up the process of diagnosing the disease, but also significantly reduces material costs. Often the doctor prescribes a comprehensive PCR analysis. The price of the examination, consisting of the determination of six pathogens, is about 1,500 rubles.

In order for the results to be reliable during the PCR study, it is necessary to pass the analysis, following the recommendations for preliminary preparation for diagnosis:

Before donating saliva, you should refrain from eating and taking medicines 4 hours before taking the material. Immediately before the procedure, rinse your mouth with boiled water.
The above rules should also be followed when taking a sample from the inner surface of the cheek. After rinsing, it is recommended to carry out a light skin massage to highlight the secret of the gland.
Urine is usually collected at home. To do this, you need to conduct a thorough toilet of the genitals. Collect 50-60 ml of urine in a sterile plastic container. To ensure the purity of the material, it is recommended for women to insert a tampon into the vagina, and for men to pull the skin fold as far as possible. You can not take the material during menstrual flow.
To donate sperm, you must refrain from sexual intercourse for 3 days before collecting the material. Doctors also advise against visiting the sauna and taking a hot bath, drinking alcohol and spicy food. 3 hours before the analysis, you need to refrain from urinating.
For delivery, for example, if a PCR test is performed for chlamydia, both women and men are recommended to have sexual rest for 3 days. 2 weeks before the analysis can not be taken antibacterial drugs. For a week, you need to stop using intimate gels, ointments, vaginal suppositories, douching. 3 hours before the examination, you must refrain from urinating. During menstruation, the sampling of material is not carried out, only 3 days after the cessation of blood discharge, you can take a urogenital smear.

PCR during pregnancy

While waiting for a baby, many sexually transmitted infections are extremely dangerous for the normal development of the fetus. STDs can provoke intrauterine growth retardation, miscarriage or premature birth, congenital malformations of the child. Therefore, it is extremely important to undergo a PCR examination in early pregnancy. It is necessary to pass the analysis when registering - up to 12 weeks.

The material is taken from the cervical canal using a special brush. The procedure is painless and does not pose a danger to the baby. Usually during pregnancy, an analysis is carried out for chlamydia by the PCR method, as well as for ureaplasmosis, mycoplasmosis, cytomegalovirus, herpes, papillomavirus. Such a complex of examinations is called PCR-6.

PCR for HIV diagnosis

Due to the fact that the method is very sensitive to changes in the body and the conditions of the diagnosis, many factors can affect the result. Therefore, PCR analysis for HIV infection is not a reliable method, its efficiency is 96-98%. In the remaining 2-4% of cases, the test gives false positive results.

But in some situations, one cannot do without PCR diagnostics of HIV. It is usually given to people with a false-negative ELISA result. Such indicators indicate that a person has not yet developed antibodies to the virus and they cannot be detected without a multiple increase in the number. This is exactly what can be achieved by conducting a blood test using the PCR method.

Such diagnostics is also necessary for children of the first year of life born from an HIV-positive mother. The method is the only way to reliably determine the status of a child.

PCR for the diagnosis of hepatitis

The polymerase chain reaction method makes it possible to detect the DNA of the hepatitis A, B, C virus long before the formation of antibodies to the infection or the onset of symptoms of the disease. The analysis of PCR for hepatitis C is especially effective, since in 85% of cases this disease is asymptomatic and, without timely treatment, passes into the chronic stage.

Timely detection of the pathogen will help to avoid complications and long-term treatment.

Comprehensive PCR examination

Comprehensive PCR analysis: examination by the polymeric chain reaction method, which includes the determination of several types of infections simultaneously: mycoplasma genitalium, mycoplasma hominis, gardnerella vaginalis, candida, Trichomonas, cytomegalovirus, herpes types 1 and 2, gonorrhea, papillomavirus. The price of such diagnostics ranges from 2000 to 3500 rubles. depending on the clinic, the materials and equipment used, as well as on the type of analysis: qualitative or quantitative. What is necessary in your case - the doctor will decide. In some cases, it is enough just to determine the presence of the pathogen, in others, for example, with HIV infection, a quantitative titer plays an important role. When diagnosing all of the above pathogens, the examination is called "PCR-12 analysis".

Deciphering the results of the analysis

Deciphering the PCR analysis is not difficult. There are only 2 scales of the indicator - "positive result" and "negative result". When a pathogen is detected, doctors can confirm the presence of the disease with 99% certainty and begin treating the patient. With a quantitative method for determining infection, the corresponding column will indicate the numerical indicator of detected bacteria. Only a doctor can determine the degree of the disease and prescribe the necessary treatment.

In some cases, for example, when determining HIV infection by PCR, with a negative result, it becomes necessary to conduct additional examinations to confirm the obtained indicators.

Where to take the analysis?

Where to take a PCR analysis: in a public clinic or in a private laboratory? Unfortunately, in municipal medical institutions, the equipment and methods are often outdated. Therefore, it is better to give preference to private laboratories with modern equipment and highly qualified personnel. In addition, in a private clinic you will get results much faster.

In Moscow, many private laboratories offer PCR analysis for various infections. For example, in such clinics as Vita, Complex Clinic, Happy Family, Uro-Pro, PCR analysis is carried out. The price of the examination is from 200 rubles. for the identification of a single pathogen.

It can be concluded that the diagnosis of infectious diseases by PCR in most cases is a fast and reliable way to detect the pathogen in the body in the early stages of infection. But still, in certain cases, it is worth choosing other diagnostic methods. Only a specialist can determine the need for such a study. Deciphering the PCR analysis also requires a professional approach. Follow your doctor's instructions and don't take tests that you don't need.

Content

Those who are interested in new diagnostic methods should find out what the PCR method is. Modern technical capabilities in the field of laboratory research provide the ability to detect many diseases on initial stages. Polymerase chain reaction (PCR) is currently considered the most accurate and new method.

PCR analysis

PCR analysis - what is it? This method uses the principles of molecular biology. To study the material, special enzymes are used that repeatedly and quickly copy DNA, RNA fragments of pathogens. Exists different types PCR analysis depending on the material being studied (blood, urine, feces, etc.). After processing, laboratory staff compare the result with the database, identify the concentration, type of pathogen.

The PCR analysis is placed in a special amplifier (device) that heats and cools the test tubes with the biomaterial. Temperature changes are needed for fragment replication. The accuracy of the result will depend on the accuracy of the temperature regime. The polymerase chain reaction method helps to identify:

Infectious mononucleosis;
cytomegalovirus infection;
viral hepatitis G, C, B, A;
sexually transmitted infections / diseases (STIs / STDs): gardnerellosis, trichomoniasis, ureaplasmosis;
herpetic infection;
oncogenic viruses;
listeriosis;
helicobacter infection;
tick-borne encephalitis, borreliosis;
tuberculosis;
candidiasis.

Blood

At the moment, due to the novelty of the technology, the PCR blood test still has a high price. For the preparation of biomaterial it is not necessary to comply with certain requirements. Even caused physical activity, stress, change in diet, changes in the composition do not affect the result of the study. PCR blood test can only spoil the reception antibacterial agents, therefore, before passing, it is necessary to pause between treatment and the test.

PCR blood test is the most common option for diagnosing chronic, acute infectious pathologies with a viral or atypical manifestation. Serological research methods have a certain difficulty in carrying out - the presence of a pathogen is determined by the presence of antibodies in the human body. The result could be false negative if the patient's condition did not give time for their development.

smear

In the field of gynecology, PCR smear analysis is used to study the presence of infectious microorganisms. Working with the material is carried out according to the same principle as with blood: a multiple increase in DNA fragments of the pathogen in order to easily identify it. It also helps to detect hidden infections in a woman. Various biological fluids can be taken for analysis: saliva, sputum, urine, blood. In gynecology, for the accuracy of the determination, a smear from the vaginal mucosa from the cervical canal is more often used.

There are certain indications for PCR. Often it needs to be done to identify an antibiotic-resistant type of pathogen. In women, the main indications for diagnosis by this method are:

a pregnancy that is difficult;
acute phase of STIs;
if there is a suspicion of the transition of STIs to the chronic stage;
search for causes of infertility.

Cala

To detect infection, a fecal PCR test may be prescribed by the doctor. In order to obtain the most reliable results after the test, it is necessary to adhere to the following rules before taking the biomaterial:

stop taking laxatives for a few days: oils, suppositories;
exclude medicines that give a specific color to the stool, for example, with iron content.

Urine

If necessary, the doctor may take urine for testing. High accuracy opens up the possibility to work with any biological fluid from which it is possible to extract virus DNA. To pass a PCR urine test, you must adhere to the following restrictions before taking the material:

at least 1 day before the procedure, stop sexual intercourse;
3 weeks before the delivery, any antibacterial treatment should be completed, because the medicines will blur the picture;
you need to take the test on an empty stomach (liquid is also prohibited);
you need to take the first morning portion of the material.

PCR test results

From the above, it is clear what PCR analysis is and the clear advantages of this research method are visible. Another plus of this diagnostic procedure is the ease of deciphering the results. Considering how much PCR analysis is done (the process itself takes about 5 hours, but the laboratory issues data in 1-2 days), this diagnostic method becomes the best option for determining many infections. Based on the results, your doctor may tell you that the test:

Negative - the studied material did not contain the desired pathogen.
Positive - RNA, DNA of the pathogen were found.

Sometimes quantitative determination of microorganisms is carried out. This is necessary for diseases that cause opportunistic pathogens. The peculiarity of these viruses is that they appear only in excess and it is extremely problematic to find them with conventional studies. This factor is important for the choice of therapeutic tactics in order to effectively treat viral infections, for example, hepatitis, HIV.

For 12 infections

To fully understand what PCR is for diagnosing infections and how effective it is, you need to know that it is capable of isolating up to 12 pathogens. The text is carried out only in laboratory conditions. For research, special enzymes are used, which increase many times the amount of RNA, DNA fragments of the virus. PCR analysis for 12 infections can reveal:

mycobacterium tuberculosis;
cytomegalovirus;
hepatitis C, G, B, A;
herpes 1, 2 types;
Epstein-Barr virus (infectious mononucleosis);
infections that are sexually transmitted by, for example, chlamydia;
listeriosis;
candida infection;
helicobacter pylori;
borreliosis, tick-borne encephalitis.

For hepatitis C

This diagnostic method helps to determine the presence of the virus in the blood. This gives doctors the opportunity to talk about its presence or absence. There are two types of PCR analysis for hepatitis C: qualitative and quantitative. The first option indicates only its presence and can be worded "detected" / "not detected". This type of test has a sensitivity of 10-500 IU/ml. This suggests that with a low content of the pathogen in the body, the analysis will be “not detected”.

Quantitative analysis is more accurate and will show the concentration of infection in the blood. This indicator is designated as "viral load", measured in the amount of viral RNA per specific volume of blood. Decryption in different laboratories may vary. In addition to measuring in IU / ml, "copy" units are used. You can recalculate copies per IU using the formula: 1 IU = 4 copies. If in the transcript the value of the presence of the virus exceeds 800,000 IU / ml (or 800 * 103), this indicates a high content of the pathogen.

For tuberculosis

The test should be done in the morning. This is important in order to prevent all the sputum that formed during the night from leaving the stomach. PCR analysis for tuberculosis is as important as ELISA, Mantoux, tomography. The test helps to identify the presence of mycobacteria, the state of urine, total immunoglobulin, ESR, and determine the state of the lungs at the moment. For the accuracy of obtaining results in the analysis of PCR, it is necessary to carry out it in compliance with the following rules:

Sowing is carried out 3 times, but complete aspiration of the contents of the stomach should be carried out only in a hospital.
Detects mycobacteria by culture of existing masses in the stomach in less than 50% of diagnoses. Even when optimal conditions are obtained, ultrasound is recommended instead.
Even with a negative result, the likelihood of developing tuberculosis with a change in ESR, immunoglobulin or other indicators cannot be completely excluded.
PCR culture is less susceptible to pathological conditions if it is obtained as part of a bronchoscopic examination, which excludes suspicion of TB in a child.

For HIV

For many people, this diagnosis is considered a death sentence. For this reason, after frequent sexual intercourse, a person becomes more attentive to the signals that his body gives (and sometimes comes up with them). The most reliable option to get confirmation or refutation this disease– PCR analysis for HIV. The test can be used to determine the following possible problems with health:

Denial/confirmation of the presence of HIV during the period of the seronegative horse.
Determination of the genotype of HIV-1, HIV-2.
Clarification of the description of the pathological process with a doubtful result of the immunoblot.
Infection after blood transfusion.
Determining HIV status in children born to carrier mothers.
Helps to establish monitoring of the viral load of the body.

For HPV

The papilloma virus can be detected in any person, for a long time it can be in a latent state. Development provokes a weakening of the immune system, stress or emotional outbursts. PCR analysis for HPV helps to determine the concentration of the virus in the blood. For this reason, it is recommended to carry out a quantitative determination rather than a qualitative one. These data will help predict the likelihood of developing a malignant nature of the infection.

The technique for diagnosing the presence of HPV is based on the main property of PCR to isolate virus DNA from the material. Due to the high sensitivity of the test, even a small amount of bacteria will be detected. Quantitative research gives doctors the opportunity to determine the degree of danger of the disease, to make a prognosis for the future. This diagnosis is mandatory for all men and women who have found themselves with warts. Quantitative PCR analysis will help determine what caused the development of HPV: a temporary decrease in immunity or a chronic disease.

For herpes

This type of diagnostics in microbiology helps to carry out PCR analysis for herpes with high accuracy. Copying of DNA fragments of the virus will occur only if the desired gene is present in the material. In this case, the test based on the results of the conduct may indicate the presence or absence of the pathogen. It will be possible to detect it even at low concentration in the blood.

Another plus of the PCR analysis is that it can detect a herpes virus infection immediately after infection, before the onset of clinical symptoms. It is possible to determine the type of herpes (1 or 2), no specific preparation is required to pass the analysis, but doctors recommend that you refuse before taking blood:

fried;
acute;
alcohol;
fatty.

During pregnancy

When carrying a child, it is very important to conduct this study in order to take into account the condition of the woman. PCR analysis during pregnancy is one of the most effective methods determining the presence of various diseases. It is necessary to conduct a test not only to identify pathologies, but also to determine the likelihood of infection of the child in utero. Only thanks to PCR diagnostics, it became possible to identify the degree of progression, the development of many infections inside the mother's womb.

Delivery of PCR analyzes

If you are wondering how the PCR analysis is taken, then each individual case should be considered, taking into account the type of biomaterial. Scraping, smear or blood sampling has its own characteristics, for example:

plasma is donated in the morning;
urine is taken only the first in the morning, under laboratory conditions in a sterile container;
a smear or scraping will be indicative only after abstinence from sexual intercourse for at least 3 days;
you can not take a smear during menstruation and 2 days after it.

Where to get tested for PCR

This type of research refers to modern and high-tech diagnostic methods. PCR tests should be taken in laboratories that have all the necessary complex to obtain full results. Qualified and trained personnel play an equally important role. Give preference to large, serious, well-known laboratories. This will help not only get the results quickly, but also ensure their reliability.

Price

Another question that is often of interest to patients is: how much does a PCR test cost? Due to the novelty of the method, the need to purchase expensive equipment, the price of the test is relatively high. The cost of PCR is affected by the type of infection for which a person will be tested. Estimated price and terms of the tests are as follows:

STIs will be checked in 1 day, the price is 400-500 rubles.
Herpes, HPV, Epstein-Barr virus, cytomeglovirus are detected per day, the price is 300-500 rubles.
An analysis for hepatitis is carried out in 5 days, the price for a qualitative option is 500 rubles, for a quantitative one - 2000 rubles.
Helicobacter pylori is detected per day, the price is 400 rubles.
Antigens, HIV antibodies, price - from 380 rubles.
Qualitative analysis of HIV RNA, price - from 3,500 rubles.
Quantitative analysis of HIV RNA, price - from 11,000 rubles.

Video

Attention! The information provided in the article is for informational purposes only. The materials of the article do not call for self-treatment. Only a qualified doctor can make a diagnosis and give recommendations for treatment, based on the individual characteristics of a particular patient.

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1. Polymerase chain reaction (PCR)

2. Principle of polymerase chain reaction method

2.1 Presence of a number of components in the reaction mixture

2.2 Temperature cycling

2.3 Basic principles of primer selection

2.4 Plateau effect

3. Stages of PCR setting

3.2 Amplification

3.4.1 Positive controls

3.4.2 Internal controls

4.1 Qualitative analysis

4.1.2 Detection of RNA molecules

3.1 Sample preparation of biological material

Different techniques are used for DNA extraction, depending on the tasks. Their essence lies in the extraction (extraction) of DNA from a biological product and the removal or neutralization of foreign impurities to obtain a DNA preparation with a purity suitable for PCR.

The method of obtaining a pure DNA preparation, described by Marmur, is considered standard and has already become classical. It includes enzymatic proteolysis followed by deproteinization and DNA reprecipitation with alcohol. This method makes it possible to obtain a pure DNA preparation. However, it is quite laborious and involves working with such aggressive and pungent substances as phenol and chloroform.

One of the currently popular methods is the DNA extraction method proposed by Boom et al. This method is based on the use of a strong chaotropic agent, guanidine thiocyanate (GuSCN), for cell lysis, and subsequent DNA sorption on a carrier (glass beads, diatomaceous earth, glass milk, etc.). After washings, DNA remains in the sample adsorbed on the carrier, from which it can be easily removed using an elution buffer. The method is convenient, technologically advanced and suitable for sample preparation for amplification. However, DNA losses are possible due to irreversible sorption on the carrier, as well as during numerous washes. This is especially important when working with small amounts of DNA in the sample. Moreover, even trace amounts of GuSCN can inhibit PCR. Therefore, when using this method, the correct choice of the sorbent and careful observance of technological nuances are very important.

Another group of sample preparation methods is based on the use of Chilex-type ion exchangers, which, unlike glass, do not adsorb DNA, but vice versa, impurities that interfere with the reaction. As a rule, this technology includes two stages: sample boiling and adsorption of impurities on an ion exchanger. The method is extremely attractive due to its simplicity of execution. In most cases, it is suitable for working with clinical material. Unfortunately, sometimes there are samples with impurities that cannot be removed using ion exchangers. In addition, some microorganisms cannot be destroyed by simple boiling. In these cases, it is necessary to introduce additional stages of sample processing.

Thus, the choice of the sample preparation method should be treated with an understanding of the purposes of the intended analyses.

3.2 Amplification

To carry out the amplification reaction, it is necessary to prepare the reaction mixture and add the analyzed DNA sample to it. In this case, it is important to take into account some features of primer annealing. The fact is that, as a rule, in the analyzed biological sample there are various DNA molecules, to which the primers used in the reaction have partial, and in some cases significant, homology. In addition, primers can anneal to each other to form primer-dimers. Both lead to a significant consumption of primers for the synthesis of side (nonspecific) reaction products and, as a result, significantly reduce the sensitivity of the system. This makes it difficult or impossible to read the results of the reaction during electrophoresis.

3.3 Evaluation of reaction results

In order to correctly assess the results of PCR, it is important to understand that this method is not quantitative. Theoretically, amplification products of single target DNA molecules can be detected by electrophoresis already after 30-35 cycles. However, in practice this is done only in cases where the reaction takes place under conditions close to ideal, which is not often encountered in life. The degree of purity of the DNA preparation has a particularly great influence on the efficiency of amplification; the presence of certain inhibitors in the reaction mixture, which in some cases can be extremely difficult to get rid of. Sometimes, due to their presence, it is not possible to amplify even tens of thousands of target DNA molecules. Thus, there is often no direct relationship between the initial amount of target DNA and the final amount of amplification products.

3.3.1 Horizontal electrophoresis method

Various methods are used to visualize the results of amplification. The most common today is the method of electrophoresis, based on the separation of DNA molecules by size. To do this, a plate of agarose gel is prepared, which is agarose frozen after melting in an electrophoretic buffer at a concentration of 1.5-2.5% with the addition of a special DNA dye, for example, ethidium bromide. The frozen agarose forms a spatial lattice. When pouring with the help of combs, special wells are formed in the gel, into which amplification products are subsequently added. The gel plate is placed in a horizontal gel electrophoresis apparatus and a constant voltage source is connected. Negatively charged DNA begins to move in the gel from minus to plus. At the same time, shorter DNA molecules move faster than long ones. The speed of DNA movement in the gel is affected by the concentration of agarose, electric field strength, temperature, the composition of the electrophoresis buffer, and, to a lesser extent, the GC composition of DNA. All molecules of the same size move at the same speed. The dye is embedded (intercalates) in planar groups into DNA molecules. After the end of electrophoresis, which lasts from 10 minutes to 1 hour, the gel is placed on the filter of a transilluminator emitting light in the ultraviolet range (254 - 310 nm). The UV energy absorbed by the DNA at 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 amplification products can be different. However, this cannot be related to the initial amount of target DNA in the sample.

3.3.2 Vertical electrophoresis method

The method of vertical electrophoresis is fundamentally similar to horizontal electrophoresis. Their difference lies in the fact that in this case polyacrylamide gels are used instead of agarose. It is carried out in a special chamber for vertical electrophoresis. Polyacrylamide gel electrophoresis has a higher resolution than agarose electrophoresis and makes it possible to distinguish DNA molecules of different sizes with an accuracy of one nucleotide. Preparation of polyacrylamide gel is somewhat more complicated than agarose. In addition, acrylamide is a toxic substance. Since the need to determine the size of the amplification product with an accuracy of 1 nucleotide rarely arises, the horizontal electrophoresis method is used in routine work.

3.4 Monitoring the progress of the amplification reaction

3.4.1 Positive controls

As a "positive control" use the DNA preparation of the desired microorganism. Non-specific amplicons differ in size from the amplicons generated by amplification with a control DNA preparation. The size of non-specific products can be either larger or smaller than the positive control. In the worst case, these dimensions may coincide and are read in electrophoresis as positive.

To control the specificity of the resulting amplification product, hybridization probes (DNA regions located within the amplifiable sequence) labeled with enzyme labels or radioactive isotopes and interacting with DNA in accordance with the same principles as primers can be used. This greatly complicates and lengthens the analysis, and its cost increases significantly.

3.4.2 Internal controls

It is necessary to control the progress of amplification in each tube with the reaction mixture. For this purpose, an additional, so-called "internal control" is used. It is any preparation of DNA that is not similar to the DNA of the desired microorganism. If the internal control is added to the reaction mixture, then it will become the same target for primer annealing as the chromosomal DNA of the desired infectious agent. The size of the internal control amplification product is selected so that it is 2 or more times larger than the amplicons generated from the amplification of the target DNA of the microorganism. As a result, if internal control DNA is introduced into the reaction mixture together with the test sample, then regardless of the presence of a microorganism in the biological sample, the internal control will cause the formation of specific amplicons, but much longer (heavier) than the amplicon of the microorganism. The presence of heavy amplicons in the reaction mixture will indicate the normal course of the amplification reaction and the absence of inhibitors. If the amplicons of the required size were not formed, but the internal control amplicons were not formed either, it can be concluded that the analyzed sample contains undesirable impurities that should be eliminated, but not the absence of the desired DNA.

Unfortunately, despite all the attractiveness of this approach, it has a significant flaw. If the required DNA is present in the reaction mixture, then the efficiency of its amplification decreases sharply due to competition with the internal control for primers. This is especially important at low concentrations of DNA in the test sample, which can lead to false negative results.

Nevertheless, provided that the problem of competition for primers is solved, this method of controlling the efficiency of amplification will certainly be very useful.

4. Methods based on the polymerase chain reaction

4.1 Qualitative analysis

The classical method of setting up PCR, the principles of which were outlined above, has been developed in some modifications aimed at overcoming the limitations of PCR and increasing the efficiency of the reaction.

4.1.1 How to set up PCR using “hot start”

To reduce the risk of formation of nonspecific products of the amplification reaction, an approach called “hot-start” is used. Its essence is to prevent the possibility of starting the reaction until the conditions in the tube are reached that ensure specific annealing of the primers.

The fact is that, depending on the HC composition and size, primers have a certain melting point (Tm). If the temperature of the system exceeds Tm, the primer is unable to adhere to the DNA strand and denatures. Under optimal conditions, i.e. annealing temperature close to the melting temperature, the primer forms a double-stranded molecule only if it is fully complementary and thus ensures the specificity of the reaction.

Exist various options"hot start" implementations:

Introducing Taq polymerase into the reaction mixture during the first cycle after heating the tube to the denaturation temperature.

Separation of the ingredients of the reaction mixture by a paraffin layer into layers (primers in the lower part, Taq polymerase and target DNA in the upper part), which are mixed when the paraffin is melted (~65-75 0 С).

Use of monoclonal antibodies to Taq polymerase. The enzyme bound by monoclonal antibodies becomes active only after the first denaturation stage, when the monoclonal antibodies irreversibly denature and release the active sites of Taq polymerase.

In all these cases, even if nonspecific annealing occurred before the onset of thermal cycling, elongation does not occur, and primer-DNA complexes are denatured upon heating, so no nonspecific products are formed. Subsequently, the temperature in the tube does not fall below the melting point, which ensures the formation of a specific amplification product.

4.1.2 Detection of RNA molecules

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. At the same time, in their life cycles there is no intermediate phase of transformation into DNA. To detect RNA, it must first be converted into the form of DNA. For this, reverse transcriptase is used, which is 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. Since at this temperature RNA molecules easily form secondary structures, the reaction efficiency decreases markedly and, according to various estimates, is approximately 5%. Attempts are being made to circumvent this drawback by using a thermostable polymerase obtained from the thermophilic microorganism Thermus Thermophilus, which exhibits transcriptase activity in the presence of Mn 2+ , as a reverse transcriptase. It is the only known enzyme capable of exhibiting both polymerase and transcriptase activity.

To carry out the reverse transcription reaction, the reaction mixture, as well as in PCR, must contain primers as a seed and a mixture of 4 dNTPs as a building material.

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

5. Organization of the technological process of setting PCR

The potentially high sensitivity of the polymerase chain reaction makes it absolutely necessary to have a particularly careful design of the PCR laboratory. This is due to the most acute problem of the method - contamination.

Contamination - getting from the external environment into the reaction mixture of specific DNA molecules that can serve as targets in the amplification reaction and give false positive results.

There are several ways to deal with this unpleasant phenomenon. One of them is the use of the enzyme N-uracil glycosylase (UG). This method is based on the ability of UG to cleave DNA molecules with embedded uracil. The amplification reaction is carried out using a dNTP mixture in which dTTP is replaced by uracil, and after thermal cycling, all amplicons formed in the tube will contain uracil. If HC is added to the reaction mixture before amplification, then the amplicons that enter the reaction mixture will be destroyed, while native DNA will remain intact and will subsequently serve as a target for amplification.

Thus, this method only to some extent eliminates the source of contamination and does not guarantee against false positive results.

Another way to deal with the results of contamination is a significant reduction in the number of reaction cycles (up to 25-30 cycles). But even with this approach, the risk of obtaining false positive results is high, because in this case, in the absence of inhibitors, it is easy to obtain an amplification product due to contamination.

Thus, despite the benefits of pre-amplification measures aimed at inactivating DNA molecules that cause false positive results, the most radical remedy is a well-thought-out organization of the laboratory.

Conclusion

The PCR method is currently the most widely used as a method for diagnosing various infectious diseases. PCR allows you to identify the etiology of the infection, even if the sample taken for analysis contains only a few DNA molecules of the pathogen. PCR is widely used in the early diagnosis of HIV infections, viral hepatitis, etc. To date, there is almost no infectious agent that cannot be detected using PCR.

Federal Agency for Education

State educational institution

Higher professional education

"Karelian State Pedagogical Academy"

Course work on the topic:

Polymerase chain reaction (PCR) and its application

Completed by: student Koryagina Valeria Alexandrovna

Checked by: Karpikova Natalya Mikhailovna

Petrozavodsk 2013

Introduction

Chapter 1 Literature Review

1.5.4 Plateau effect

1.5.6 Amplification

Conclusion

Introduction

The last twenty years have been marked by the widespread introduction of molecular genetic methods into the biological, medical, and agricultural sciences.

By the early 1970s, it seemed that molecular biology had reached a certain degree of perfection. During this period, microorganisms were the main object of molecular genetic research. The transition to eukaryotes presented researchers with completely new problems that could not be solved using the methods that existed at that time. genetic analysis. A breakthrough in the development of molecular genetics became possible due to the emergence of a new experimental tool - restriction endonucleases. In subsequent years, the number of direct DNA analysis methods based on qualitatively different approaches began to increase rapidly.

Modern technologies in many cases, they made it possible to start studying the fine structural and functional organization of the nuclear and extranuclear genomes of various organisms at a deeper level. This was of particular importance for the development of new methods of diagnosis and treatment. various diseases. No less important was the possibility of using the achievements of molecular genetics in population biology and breeding to identify and analyze the genetic variability of populations, varieties and strains, identify and certify economically valuable individuals, create genetically modified organisms, and solve other issues.

Each method has its own advantages and disadvantages. There is no universal method that could solve all the problems that arise. Therefore, the choice of a specific method for the ongoing research is one of the most important stages of any scientific work.

Chapter 1 Literature Review

1.1 History of the discovery of the polymerase chain reaction (PCR)

In 1983 K.B. Mullis et al. published and patented the polymerase chain reaction (PCR) method, which was destined to have a profound impact on all areas of research and application of nucleic acids. The significance of this method for molecular biology and genetics turned out to be so great and obvious that seven years later the author was awarded the Nobel Prize in Chemistry.

At the beginning of using the method, after each heating-cooling cycle, DNA polymerase had to be added to the reaction mixture, since it was inactivated at the high temperature necessary to separate the DNA helix chains. The reaction procedure was relatively inefficient, requiring a lot of time and enzyme. In 1986, the polymerase chain reaction method was significantly improved. It has been proposed to use DNA polymerases from thermophilic bacteria. These enzymes proved to be thermostable and were able to withstand many reaction cycles. Their use made it possible to simplify and automate PCR. One of the first thermostable DNA polymerases was isolated from bacteria Thermus aquaticusand named Taq-polymerase.

The possibility of amplifying any DNA segment, the nucleotide sequence of which is known, and obtaining it after the completion of PCR in a homogeneous form and in a preparative amount make PCR alternative method molecular cloning of short DNA fragments. In this case, there is no need to apply complex methodological techniques that are used in genetic engineering in conventional cloning. The development of the PCR method has greatly expanded the methodological possibilities of molecular genetics, and, in particular, genetic engineering, so much so that it has radically changed and strengthened the scientific potential of many of its areas.

1.2 Varieties of polymerase chain reaction (PCR)

· Nested PCR- used to reduce the number of by-products of the reaction. Use two pairs of primers and carry out two consecutive reactions. The second pair of primers amplifies the DNA region within the product of the first reaction.

· Inverted PCR- is used when only a small area within the desired sequence is known. This method is especially useful when it is necessary to determine neighboring sequences after DNA has been inserted into the genome. For the implementation of inverted PCR, a series of DNA cuts is carried out with restriction enzymes<#"justify">polymerase chain reaction primer

· Group-specific PCR- PCR for relatives<#"center">1.3 Polymerase chain reaction

Discovered in the mid-1980s, the polymerase chain reaction (PCR) can increase the number of copies of an original sample millions of times within a few hours. During each cycle of the reaction, two copies are formed from the original molecule. Each of the synthesized DNA copies can serve as a template for the synthesis of new DNA copies in the next cycle. In this way, multiple repetition cycles, leads to an exponential increase in the number of copies. It follows from the calculations that even if there are 30 cycles, the number of copies of the original molecule will be more than 1 billion. Even if we take into account that not all amplicons are duplicated during each cycle, the total number of copies, despite this, is quite a large figure.

Each cycle of the polymerase chain reaction (PCR) consists of the following steps:

· Denaturation - An increase in temperature causes a double-stranded DNA molecule to unwind and split into two single-stranded ones;

· Annealing - Lowering the temperature allows primers to attach to complementary regions of the DNA molecule;

· Elongation - The enzyme DNA polymerase completes the complementary strand.

For amplification of the selected fragment, two oligonucleotide primers (seeds) flanking a certain DNA region are used. Primers oriented 3 - ends towards each other and in the direction of the sequence that needs to be amplified. DNA polymerase carries out the synthesis (completion) of mutually complementary DNA chains, starting with primers. During DNA synthesis, primers are physically inserted into the chain of newly synthesized DNA molecules. Each strand of the DNA molecule formed using one of the primers can serve as a template for the synthesis of a complementary DNA strand using the other primer.

1.4 Conducting a polymerase chain reaction (PCR)

The polymerase chain reaction is carried out in special thin-walled polypropylene test tubes, compatible in size with the used thermal cycler (amplifier) - a device that controls the temperature and time characteristics of the stages of the polymerase chain reaction (PCR).

1.5 Principle of the polymerase chain reaction method

Polymerase chain reaction (PCR) is an in vitro DNA amplification method that can isolate and multiply a specific DNA sequence billions of times within a few hours. The ability to obtain a huge number of copies of one strictly defined region of the genome greatly simplifies the study of an existing DNA sample.

To carry out a polymerase chain reaction, a number of conditions must be met:

1.5.1 Presence of a number of components in the reaction mixture

The main components of the reaction (PCR) mixture are: Tris-HCl, KCl, MgCl 2, a mixture of nucleotide triphosphates (ATP, GTP, CTP, TTP), primers (oligonucleotides), analyzed DNA preparation, thermostable DNA polymerase. Each of the components of the reaction mixture is directly involved in the polymerase chain reaction (PCR), and the concentration of reagents directly affects the course of amplification.

· Tris-HCl - determines the pH of the reaction mixture, creates a buffer capacity. The activity of DNA polymerase depends on the pH of the medium, so the value of the pH directly affects the course of the polymerase chain reaction. Usually the pH value is in the range of 8 - 9.5. The high pH is due to the fact that as the temperature rises, the pH of the Tril-HCl buffer drops.

· KCl - the concentration of potassium chloride up to 50 mm affects the course of the processes of denaturation and annealing, the concentration above 50 mm inhibits DNA polymerase.

· MgCl 2- because DNA polymerase is Mg 2+- dependent enzyme, then the concentration of magnesium ions affects the activity of the enzyme (Mg 2+forms complexes with NTP - it is these complexes that are the substrate for polymerase). A high concentration leads to an increase in nonspecific amplification, and a low one leads to inhibition of the reaction, the optimum (for various polymerases) is in the region of 0.5 - 5 mM. In addition, the concentration of magnesium salts affects the course of denaturation and annealing processes - an increase in the concentration of Mg 2+causes an increase in the melting temperature of DNA (i.e., the temperature at which 50% of double-stranded DNA strands are broken into single-stranded strands).

· NTP - nucleotide triphosphates are direct monomers of nucleic acids. To prevent chain termination, an equal ratio of all four nucleotide triphosphates is recommended. The low concentration of these components in the reaction mixture increases the probability of errors in the construction of the complementary DNA strand.

· Primers - The most optimal is the use of primers with a melting point difference of no more than 2 - 4 o C. Sometimes during long-term storage at a temperature of 4 o With, or after a large number of freezing - thawing, the primers form secondary structures - dimers, reducing the efficiency of the PCR. The elimination of this problem is reduced to incubation in a water bath (T=95 o C) for 3 minutes and subsequent rapid cooling to 0o FROM.

· DNA preparations - the quantity and quality of the DNA preparation (matrix) directly affects the course and parameters of the polymerase chain reaction. Excess DNA sample inhibits the polymerase chain reaction (PCR). Impurities of various substances in the DNA preparation can also reduce the efficiency of the polymerase chain reaction (PCR): sodium acetate, sodium chloride, isopropanol, ethanol, heparin, phenol, urea, hemoglobin, etc.

· DNA polymerase - when using a small amount of DNA polymerase, a decrease in the synthesis of the final product is observed in direct proportion to the size of the fragments. An excess of polymerase by 2–4 times leads to the appearance of diffuse spectra, and by 4–16 times, low molecular weight nonspecific spectra. The range of concentrations used is 0.5 - 1.5 units of activity in terms of 25 µl of the PCR mixture.

In addition to the main components of the PCR mixture, a number of additional substances are used that improve the qualitative and quantitative indicators of PCR: acetamide (5%) - an increase in the solubility of the main components; betaine (sodium salt) - stabilization of DNA polymerase, lowering the melting point of DNA, equalizing the melting point; bovine albumin (10-100 μg / ml) - stabilization of DNA polymerase; dimethyl sulfoxide (1-10%) - increasing the solubility of the main components; formamide (2-10%) - an increase in the specificity of annealing; glycerol (15-20%) - an increase in the thermal stability of the enzyme, a decrease in the temperature of denaturation of a DNA sample; ammonium sulfate - lowering the temperature of denaturation and annealing.

1.5.2 Cycle and temperature

General form polymerase chain reaction (PCR) programs are as follows:

stage. Prolonged primary denaturation of the DNA preparation.1 cycle

stage. Rapid denaturation of the DNA preparation. Primer annealing. Elongation.30 - 45 cycles.

stage. Prolonged elongation. Cooling of the reaction mixture. 1 cycle.

Each element of the stage - denaturation, annealing, elongation - has individual temperature and time characteristics. The parameters of temperature and flow time of each element are selected empirically, in accordance with the qualitative and quantitative indicators of the amplification products.

Denaturation. During this element of the polymerase chain reaction, a double-stranded DNA molecule is split into two single-stranded ones. Temperature parameters of denaturation are in the range of 90 - 95 o C, but in the case of a DNA sample with a high content of guanine and cytosine, the temperature should be increased to 98 o C. The temperature of denaturation should be sufficient to completely denature - cleave the DNA strands and avoid "sudden cooling" or rapid annealing, however, thermostable DNA polymerase is less stable at high temperatures. Thus, the selection of optimal denaturation temperature parameters for the primer/sample ratio (DNA preparation) is an important condition for amplification. If the denaturation temperature in the first step is above 95 o C, it is recommended to add DNA polymerase to the reaction mixture after primary denaturation. The duration of this element of the stage during the polymerase chain reaction (PCR) should be sufficient for complete DNA denaturation, but at the same time not significantly affect the activity of DNA polymerase at a given temperature.

Annealing. Annealing temperature (T a ) is one of the most important parameters of the polymerase chain reaction. The annealing temperature for each specific primer is selected individually. It depends on the length and nucleotide composition of the primer. Usually it is lower by 2 - 4 o From the melting point value (T m ) primer. If the annealing temperature of the system is below the optimum, then the number of nonspecific amplified fragments increases and, conversely, more heat reduces the amount of amplified products. In this case, the concentration of specific amplicons can sharply decrease, up to inhibition of the polymerase chain reaction (PCR). Increasing the annealing time also leads to an increase in the number of nonspecific amplicons.

Elongation. Typically, each type of thermostable DNA polymerase has an individual temperature optimum of activity. The rate of synthesis of a complementary DNA strand by an enzyme is also a value specific to each polymerase (on average, it is 30–60 nucleotides per second, or 1–2 thousand bases per minute), so the elongation time is selected depending on the type of DNA polymerase and the length of the amplified region.

1.5.3 Basic principles of primer selection

When creating a PCR test system, one of the main tasks is the correct selection of primers that must meet a number of criteria:

Primers must be specific. Particular attention is paid to 3 - the ends of the primers, since it is from them that Taq polymerase begins to complete the complementary DNA chain. If their specificity is insufficient, then it is likely that undesirable processes will occur in the test tube with the reaction mixture, namely, the synthesis of nonspecific DNA (short or long fragments). It is visible on electrophoresis in the form of heavy or light additional bands. This makes it difficult to evaluate the results of the reaction, since it is easy to confuse a specific amplification product with synthesized foreign DNA. Part of the primers and dNTPs is consumed for the synthesis of nonspecific DNA, which leads to a significant loss of sensitivity.

Primers should not form dimers and loops, i.e. no stable double strands should be formed by annealing the primers to themselves or to each other.

1.5.4 Plateau effect

It should be noted that the process of accumulation of specific amplification products exponentially takes only a limited time, and then its efficiency drops critically. This is due to the so-called "plateau" effect.

term effect plateau used to describe the process of accumulation of PCR products in the last cycles of amplification.

Depending on the conditions and the number of cycles of the amplification reaction, at the time the effect is achieved plateau the utilization of substrates (dNTPs and primers), the stability of reactants (dNTPs and enzyme), the amount of inhibitors, including pyrophosphates and DNA duplexes, competition for reactants with non-specific products or primer-dimers, the concentration of a specific product, and incomplete denaturation at high concentrations of amplification products.

The lower the initial concentration of the target DNA, the higher the risk of the reaction plateau". This point can occur before the number of specific amplification products is sufficient to be analyzed. Only well-optimized test systems can avoid this.

1.5.5 Sample preparation of biological material

Thus, the choice of the sample preparation method should be treated with an understanding of the purposes of the intended analyses.

1.5.6 Amplification

1.6 Composition of the standard PCR reaction mixture

x PCR buffer (100 mM Tris-HCl solution, pH 9.0, 500 mM KCl solution, 25 mM MgCl2 solution ) …….2.5 µl

Water (MilliQ) ……………………………………………………….18.8 µl

A mixture of nucleotide triphosphates (dNTPs)

mM solution of each………………………………………….……….0.5 µl

Primer 1 (10 mM solution) ………………………………………….….1 µl

Primer 2 (10 mM solution) ………………………………………….….1 µl

DNA polymerase (5 units / µl) ……………………………………………0.2 µl

DNA sample (20 ng/µl) …………………………………………..1 µl

1.7 Evaluation of reaction results

Chapter 2: Applications of the Polymerase Chain Reaction

PCR is used in many areas for analysis and in scientific experiments.

Criminalistics

PCR is used to compare so-called "genetic fingerprints". We need a sample of genetic material from the crime scene - blood, saliva, semen, hair, etc. It is compared with the suspect's genetic material. A very small amount of DNA is enough, theoretically - one copy. The DNA is cut into fragments, then amplified by PCR. The fragments are separated by DNA electrophoresis. The resulting picture of the location of the DNA bands is called the genetic fingerprint.

Establishing paternity

Results of electrophoresis of DNA fragments amplified by PCR. Father. Child. Mother. The child inherited some features of the genetic imprint of both parents, which gave a new, unique imprint.

Although "genetic fingerprints" are unique, family ties can still be established by making several such fingerprints. The same method can be applied, with slight modifications, to establish evolutionary relationships among organisms.

Medical diagnostics

PCR makes it possible to significantly speed up and facilitate the diagnosis of hereditary and viral diseases. The gene of interest is amplified by PCR using appropriate primers and then sequenced to determine mutations. Viral infections can be detected immediately after infection, weeks or months before symptoms of the disease appear.

Personalized medicine

Sometimes drugs are toxic or allergenic for some patients. The reasons for this are partly in individual differences in the susceptibility and metabolism of drugs and their derivatives. These differences are determined at the genetic level. For example, in one patient, a certain cytochrome may be more active, in another - less. In order to determine what kind of cytochrome a given patient has, it is proposed to perform a PCR analysis before using the drug. This analysis is called preliminary genotyping.

Gene cloning

Gene cloning is the process of isolating genes and, as a result of genetic engineering manipulations, obtaining a large amount of the product of a given gene. PCR is used to amplify a gene, which is then inserted into a vector, a piece of DNA that carries the foreign gene into the same organism or another organism that is easy to grow. As vectors, for example, plasmids or viral DNA are used. The insertion of genes into a foreign organism is usually used to obtain a product of this gene - RNA or, most often, a protein. In this way, many proteins are obtained in industrial quantities for use in agriculture, medicine, etc.

DNA sequencing

In the sequencing method using fluorescently or radioactively labeled dideoxynucleotides, PCR is an integral part, since it is during polymerization that nucleotide derivatives labeled with a fluorescent or radioactive label are inserted into the DNA chain. This stops the reaction, allowing the positions of specific nucleotides to be determined after separation of the synthesized strands in the gel.

Mutagenesis

Currently, PCR has become the main method of mutagenesis. The use of PCR made it possible to simplify and speed up the mutagenesis procedure, as well as to make it more reliable and reproducible.

The PCR method made it possible to analyze the presence of human papillomavirus sequences in sections of biopsies of human cervical neoplasms embedded in paraffin 40 years before this study. Moreover, with the help of PCR, it was possible to amplify and clone fragments of mitochondrial DNA from the fossil remains of the human brain of the age of 7 thousand years!

On lysates of individual human spermatozoa, the possibility of simultaneously analyzing two loci located on different nonhomologous chromosomes was demonstrated. This approach provides a unique opportunity for fine genetic analysis and the study of chromosomal recombination, DNA polymorphism, etc. The method of analyzing individual spermatozoa immediately found practical use in forensic medicine, since HLA typing of haploid cells allows determining paternity or identifying a criminal (the HLA complex is a set of human major histocompatibility complex genes; the loci of the HLA complex are the most polymorphic of all known in higher vertebrates: within a species, at each locus there is an unusual a large number of different alleles - alternative forms of the same gene).

Using PCR, it is possible to identify the correctness of the integration of foreign genetic structures in a predetermined region of the genome of the studied cells. Total cellular DNA is annealed with two oligonucleotide primers, one of which is complementary to the site of the host DNA near the insertion point, and the other to the sequence of the integrated fragment in the antiparallel DNA strand. The polymerase chain reaction in the case of an unchanged chromosomal DNA structure at the proposed insertion site leads to the formation of single-stranded DNA fragments of an indefinite size, and in the case of a planned insertion, double-stranded DNA fragments of a known size, determined by the distance between the annealing sites of the two primers. Moreover, the degree of amplification of the analyzed region of the genome in the first case will be linearly dependent on the number of cycles, and in the second - exponentially. The exponential accumulation during PCR of an amplified fragment of a predetermined size makes it possible to visually observe it after electrophoretic fractionation of a DNA preparation and make an unambiguous conclusion about the insertion of a foreign sequence into a given region of chromosomal DNA.

Conclusion

List of used literature

1.Padutov V.E., Baranov O.Yu., Voropaev E.V. Methods of molecular - genetic analysis. - Minsk: Unipol, 2007. - 176 p.

2.PCR "in real time" / Rebrikov D.V., Samatov G.A., Trofimov D.Yu. and etc.; ed. b. n. D.V. Rebrikov; foreword L.A. Osterman and acad. RAS and RAAS E.D. Sverdlov; 2nd ed., rev. and additional - M.: BINOM. Knowledge Laboratory, 2009. - 223 p.

.Patrushev L.I. Artificial genetic systems. - M.: Nauka, 2005. - In 2 tons

.B. Glick, J. Pasternak Molecular biotechnology. Principles and application 589 pages, 2002

5.Shchelkunov S.N. genetic engineering. - Novosibirsk: Sib. univ. publishing house, 2004. - 496 p.

Edited by A.A. Vorbyeva "Polymerase chain reaction and its application for diagnostics in dermatovenereology"; Medical News Agency - 72 pages

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