Key stages of molecular-genetic analysis
Any study of genetic material is built upon a specific algorithm that allows obtaining accurate data from a minimal volume of biological material. The process can be divided into two key steps:
- Sample collection. This is a mandatory initial step of the work. Specialists isolate nucleic acids (DNA or RNA) from the provided biological material.
- Amplification. Since making a diagnosis usually does not require analyzing the entire genome, only a small segment of it is investigated. For equipment to recognize this fragment, it needs to be "amplified." Amplification is a multiplication process that yields a sufficient number of copies of the target genome fragment for subsequent analysis.
Polymerase chain reaction (PCR)
One of the primary technologies in molecular genetics is polymerase chain reaction (PCR). This is a specific method of amplification, i.e., targeted replication of a given DNA segment.
A crucial condition for PCR is that the process is carried out strictly in vitro (in a test tube, outside a living cell). The technology boasts incredible efficiency: in just a few hours, the number of copies of a specific DNA sequence increases millions of times.
The main advantage of the method lies in its striking sensitivity. An extremely negligible amount of initial genetic material is required to initiate the reaction and obtain a reliable result.
Diagnosis of hereditary pathologies
Molecular-genetic technologies are actively used to identify various hereditary diseases. The choice of a specific approach depends on the nature of the targeted pathology.
- Gene sequencing (determining the exact nucleotide sequence). Used for diagnosing hemophilias, thrombophilias, various hemoglobinopathies, as well as mitochondrial diseases.
- Direct detection of mutant genes. The method of choice when suspecting cystic fibrosis, phenylketonuria, Duchenne muscular dystrophy, and fragile X syndrome.
- Genetic analysis of DNA polymorphism (studying gene linkage in parents and child). This technology allows diagnosing about 300 different hereditary diseases, including all the aforementioned pathologies.
- Determination of the primary gene product. This approach is used to identify Duchenne muscular dystrophy and specific storage diseases (lysosomal and peroxisomal disorders).
Other molecular approaches
In addition to basic sequencing and direct detection, geneticists have other specific tools in their arsenal. One of them is RFLP analysis (restriction fragment length polymorphism).
The essence of the RFLP method is targeted cleavage of the DNA molecule into fragments using special enzymes—restriction endonucleases. Differences in the length of the resulting fragments allow drawing conclusions about the presence or absence of genetic polymorphisms and mutations in the patient.
Briefly: answers to topical questions
What temperature stages (cycles) does a classical polymerase chain reaction (PCR) include?
A classical polymerase chain reaction includes three sequential stages differing in temperature regime:
- Denaturation — heating to 92–95 °C (according to some sources, 94 °C). Destruction of the double-stranded DNA structure and strand separation occur.
- Annealing — cooling to 37–60 °C (or 52–60 °C). At this stage, primers bind complementarily to the template DNA strands.
- Elongation (polymerization) — heating to 63–75 °C (or 72 °C). Synthesis of new strands using thermostable DNA polymerase.
What reagents and components are strictly required to perform PCR in vitro?
To perform polymerase chain reaction in vitro, the following components are required:
- DNA template — the starting DNA target.
- Primers — short primer oligonucleotides complementary to regions of the template DNA.
- Thermostable DNA polymerase (Taq polymerase) — the enzyme for synthesizing the new strand.
- Free nucleotides — used for the synthesis of new DNA strands.
For the diagnosis of which specific diseases is gene linkage analysis (genetic analysis of DNA polymorphism) used?
Genetic analysis of DNA polymorphism (gene linkage) is used to diagnose about 300 hereditary diseases. Among them:
- Hemophilias, thrombophilias, hemoglobinopathies, mitochondrial diseases.
- Cystic fibrosis, fragile X syndrome, phenylketonuria, Duchenne muscular dystrophy.
Class and exam questions
What does the initial step of any molecular-genetic study consist of?
The very first step is always the isolation and collection of nucleic acid samples (DNA or RNA) from the provided biological material.
What is DNA amplification needed for?
Amplification is necessary to multiply the required genome fragment. Only a small stretch of DNA is needed for diagnostics, but for equipment to analyze it, a sufficient number of its copies must be generated.
Under what conditions does the polymerase chain reaction take place?
PCR is a DNA amplification method performed exclusively in artificial conditions, i.e., in vitro.
What diseases can be detected using direct detection of mutant genes?
This approach is used to diagnose cystic fibrosis, phenylketonuria, Duchenne muscular dystrophy, and fragile X syndrome.
What else to study on this topic
- Mechanisms of primary sequence determination (sequencing)
- Principles of direct detection of mutant genes
- Genetic analysis of DNA polymorphism (gene linkage)
- RFLP technology and the action of restriction endonucleases
- Methods for determining the primary gene product
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