Laboratory Results: Polymerase Chain Reaction (PCR)

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Laboratory Results: Polymerase Chain Reaction (PCR)
Laboratory Results: Polymerase Chain Reaction (PCR)
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Laboratory results: polymerase chain reaction (PCR)

The polymerase chain reaction (PCR) is the most important laboratory method for examining the fine molecular structure of genetic material. This is made up of deoxyribonucleic acid (DNA), which builds up the genetic code of humans, but also of animals and plants.

In human medicine, PCR is used to clarify hereditary diseases and genetic issues (risk of disease, paternity test, etc.), but also in the diagnosis of numerous infectious diseases. In contrast to serological diagnostics (determination of antibodies against pathogens in the blood), PCR methods represent a direct laboratory medical detection method for the clarification of infectious diseases.

On the one hand, the advantage of PCR is the rapid availability of the test results. In addition, the PCR methods are usually very sensitive.

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  • What is the polymerase chain reaction (PCR) and what is it investigated?
  • How does the PCR work?
  • What test results does the PCR provide?
  • Are there any further developments in conventional PCR?
  • What are the main areas of application for PCR?
  • Are there reference values ​​for PCR test results?

This means that even the smallest amounts of genetic material of a pathogen (bacteria, viruses, etc.) lead to a reliably positive result within the detection period of the respective pathogen in the corresponding test material. Primarily blood, but also other body fluids (sputum, urine, liquor, etc.) are used as examination materials for PCR methods.

What is the polymerase chain reaction (PCR) and what is it investigated?

The polymerase chain reaction ("Polymerase Chain Reaction" - PCR) is the most important laboratory method for examining the fine molecular structure of genetic material. For this reason, this investigation method is also called so-called

“Molecular diagnostics”

The basis of the genetic material (also called "genetic material") is a special, long-chain molecule:

the "DNA" (deoxyribonucleic acid)

In humans, the genetic material made up of double-stranded DNA (two complementary strands) is located in the nucleus of all body cells, whereby all body cells of an individual each have identical genetic material. In addition, the exact composition of each person's individual genetic makeup is unique - comparable to the fingerprint, which is also unique for each person.

For this reason, the genetic material is also called

"Genetic code" is called because the blueprint of all body structures of a person is mapped in this encrypted form and saved in this way and can be passed on

Within the cell nuclei of the body's cells, the DNA is in a special order known as

"Chromosomes" (hereditary bodies) is called. In humans, these always occur in pairs in the body cells, one being inherited from the mother and the other from the father. In this sense, all body cells of a person contain a total of 46 chromosomes - 22 pairs of so-called autosomes and one pair of so-called sex chromosomes

With the help of the laboratory method of PCR, on the one hand, the fine structure of human DNA can be examined, which is important for diagnosing diseases or for clarifying specific questions:

  • Clarification of hereditary diseases,
  • Assessment of disease risk,
  • Investigation of innate peculiarities of metabolism,
  • forensic (so-called "forensic") analysis (e.g. proof of paternity) and much more

On the other hand, not only does humans have a corresponding genetic make-up, but actually all life forms on earth have their own specific genetic make-up:

  • Animals,
  • Plants,
  • Mushrooms,
  • Bacteria,
  • Viruses,
  • Parasites etc.

The genetic makeup of these life forms also consists mainly of DNA or, in the case of some viruses, also of RNA (ribonucleic acid), which, in contrast to DNA, is single-stranded. In medical diagnostics, PCR is therefore also used to clarify numerous infectious diseases:

  • bacterial infections (e.g. tuberculosis, bacterial sexually transmitted diseases),
  • viral infections (e.g. viral hepatitis, HIV infection),
  • parasitic infections (e.g. malaria) and much more

How does the PCR work?

The PCR method was developed in 1983 by the American biochemist Kary B. Mullis and is essentially based on two principles:

  • Multiplication ("amplification") of a small part of the genetic material (DNA or RNA) as well
  • Detection and identification of the multiplied ("amplified") products of the amplification.

The following components are therefore required to carry out a PCR:

  • a test material (e.g. blood, cell or tissue samples),
  • certain reagents ("polymerase", "primer", "nucleotides" [these are the DNA building blocks] etc.) and laboratory equipment for amplifying the genetic material as well
  • a detection system for the PCR products.

In terms of the reagents and laboratory equipment needed to perform a PCR, the following enzyme is at the heart of the entire PCR process:

the Taq polymerase

This is an enzyme that is obtained from the microorganism "Thermus aquarius" (Taq) - a bacterial strain that exists in the vicinity of hot, volcanic springs and can survive adapted to the high temperatures of its environment.

The specialty of Taq polymerase is that this enzyme can also fulfill its function at high temperatures above 50 ° Celsius (C) - namely

the multiplication ("amplification") of DNA

In addition to the Taq polymerase, it must be precisely defined in advance of the PCR analysis which part of the DNA is to be examined. For this purpose, short DNA fragments are required, the fine structure of which corresponds to the DNA target region with regard to the respective genetic code (“DNA sequence). These DNA fragments are called

"Primer" called

The actual amplification process in the context of a PCR finally comprises the following steps:

  • Extraction of the genetic material (DNA or RNA) from the test material.

    When examining RNA, the ribonucleic acid code sequence must first be converted into a DNA code sequence, which must be done with the enzyme "reverse transcriptase" in an analytical step before the actual amplification

  • Amplification of the genetic material in several steps ("cycles"):

    • Step one - "DNA denaturation": by heating (95 ° C) the double-stranded DNA, it is melted into single strands.
    • Step two - “Annealing”: at a lower temperature (approx. 55 ° C) the “primers” can now attach to their DNA target sequences.
    • Step three - “Elongation”: at a temperature of 72 ° C the enzyme Taq polymerase now extends (“elongates”) the primer start sequence using so-called “nucleotides” (these are the DNA building blocks) and builds it up this way a new, elongated DNA molecule.

Since steps one to two are repeated in several cycles, longer and longer newly formed DNA chains are created, which is where the name of this laboratory process “polymerase chain reaction” comes from.

  • Analysis of the newly created DNA chains (so-called "PCR products"):

    At the end of the chain reaction (cycles), the PCR products - ie the newly created DNA molecules - are examined and evaluated qualitatively or quantitatively. There are numerous possibilities for this evaluation itself, mostly based on staining (especially fluorescent dyes) of the DNA and making it visible (by means of gel electrophoresis or photometric fluorescence detection)

What test results does the PCR provide?

The results of modern PCR methods can be summarized in two groups:

  • qualitative and
  • quantitative results.

In qualitative PCR, it is examined whether a certain segment of the genetic material (DNA or RNA) is present in the test material (“positive” result) or not (“negative” result).

Qualitative PCR is therefore mainly used to clarify the following diagnostic questions ("yes / no" results):

  • Diagnosis of hereditary diseases,
  • Clarification of mutations,
  • Recognition of genetic characteristics (e.g. predisposition for certain diseases) etc.

The quantitative PCR is a further development of this laboratory method, whereby not only the presence of a certain area of ​​the genetic material is detected, but also the amount of genetic material at the same time. Accordingly, quantitative PCR is mainly used in the following areas of medicine:

  • As part of the infection diagnosis, the amount of pathogens in the patient's body can be precisely determined. In the case of virus infections, this PCR method is used to treat the so-called

    Viral load examined

In addition, quantitative PCR is also very important in basic research, whereby not only human medicine, but also a number of other scientific areas (veterinary medicine, biochemistry, biology, etc.) benefit from this process.

The advantage of PCR in the diagnosis of infectious diseases is that the test results of this laboratory method are available extremely quickly (usually within one working day). In addition, the PCR is a highly sensitive laboratory method. This means that even the smallest amounts of bacteria or viruses in the test material lead to a reliably positive result.

Are there any further developments in conventional PCR?

A special further development of the PCR methodology is the

Multiplex PCR

In multiplex PCR, several DNA target sequences (read: several primers) are used at the same time. This creates a mixture of PCR products that have to be detected and evaluated accordingly.

Multiplex PCR is also primarily used in medicine

  • human genetics as well
  • the infection diagnosis.

The advantage of this method is that overall less test material has to be obtained from the patients and a series of test results can be created in one analysis step. However, these advantages with regard to diagnostics are offset by the high price for analysis in many cases.

Another specialty for the use of PCR is the laboratory process of

DNA sequencing

This is a diagnostic possibility to decipher the exact sequence of the genetic code in a patient. The sequencing method can also be based on PCR technology.

However, since sequencing is considerably more complex and cost-intensive than conventional PCR methods, it is currently reserved for special diagnostic (extended genome analyzes) and scientific questions.

What are the main areas of application for PCR?

With regard to the areas of application of PCR in human medicine, this laboratory procedure is used for the following diagnostic questions:

  • Clarification of hereditary diseases - there are already PCR methods for more than 600 different hereditary diseases.
  • Pharmacogenetics - by studying genetic characteristics of liver metabolism, conclusions can be drawn about the effectiveness and dosage of drugs.
  • Tumor medicine ("oncology") - here, on the one hand, PCR is used to clarify risk factors for certain tumor diseases (eg breast cancer). On the other hand, cells and tissues in tumors that have already occurred can also be examined with regard to diverse mutations, which is playing an increasingly important role in the treatment of these diseases in particular.
  • Forensic medicine ("forensics") - here the PCR is of great importance in, for example, paternity proceedings and in criminology ("DNA fingerprinting").
  • Infection medicine - PCR has an indispensable value in the diagnosis, progression and therapy control of bacterial, viral and parasitic infectious diseases.

For more information on cancer, see Cancer.

Are there reference values ​​for PCR test results?

With regard to reference values ​​in PCR methods, the respective diagnostic question must always be taken into account. Since certain genetic features are detected as part of an investigation for hereditary diseases (e.g. mutation analysis), there is no reference value (“positive” or “negative”) in such laboratory analyzes, just as there is no reference value for hair color, for example. Because hair color is also a genetic characteristic.

In addition, laboratory analyzes for innate hereditary traits are subject to strict regulations in this country, for which the provisions of the Austrian Genetic Engineering Act (GTG) are authoritative (determination of an existing disease according to Section 65 GTG). As a result, this analysis may only be carried out by the medical laboratory after written confirmation has been received from the person to be examined, the legal guardian (in the case of minors who are incapable of making a decision) or the legal representative (in the case of persons of age who are incapable of making decisions) about the comprehensive information provided by attending physician must be carried out with regard to this genetic test (Section 69 GTG).

Furthermore, patients generally have the right to prohibit genetic tests from being carried out on themselves.

For non-human genetic PCR laboratory analyzes, however, much less strict regulations apply. Mostly it concerns laboratory methods from the field of infection diagnosis.

With regard to the reference values ​​of these laboratory methods, a distinction must be made between qualitative and quantitative methods:

Qualitative PCR methods usually deliver a “positive” or “negative” result, whereby “negative” is usually the reference value

With the quantitative PCR method, depending on the method, appropriate threshold values ​​(so-called "cut-offs") are defined, from when a result is to be assessed as pathological.

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