Molecular biological research methods play a big role in modern medicine, forensics and biology. Thanks to advances in the study of DNA and RNA, a person is able to study the genome of an organism, determine the causative agent of a disease, recognize the desired nucleic acid in a mixture of acids, etc.
Molecular biological research methods. What is it?
Back in the 70s and 80s, scientists for the first time succeeded in deciphering the human genome. This event gave impetus to the development of genetic engineering and molecular biology. The study of the properties of DNA and RNA has led to the fact that it is now possible to use these nucleic acids for the purpose of diagnosing a disease, studying genes.
Obtaining DNA and RNA
Molecular biological diagnostic methods require the presence of the starting material: more often it is nucleic acids. There are several ways to isolate these substances from the cells of living organisms. Each of them has its own advantages and disadvantages, and it is necessarytake into account when choosing a method for isolating pure nucleic acids.
1. Obtaining DNA according to Marmur. The method consists in treating a mixture of substances with alcohol, as a result of which pure DNA precipitates. The disadvantage of this method is the use of aggressive substances: phenol and chloroform.
2. Isolation of DNA according to Boom. The main substance used here is guanidine thiocyanate (GuSCN). It contributes to the precipitation of deoxyribonucleic acid on specialized substrates, from which it can subsequently be collected using a special buffer. However, GuSCN is an inhibitor of PTC, and even a small part of it that gets into the precipitated DNA can affect the course of the polymerase chain reaction, which plays an important role in working with nucleic acids.
3. Sedimentation of impurities. The method differs from the previous ones in that it is not the molecules of deoxyribonucleic acid that are precipitated, but impurities. To do this, ion exchangers are used. The disadvantage is that not all substances can precipitate.
4. Mass screening. This method is used in cases where exact information about the composition of the DNA molecule is not needed, but it is necessary to obtain some statistical data. This is explained by the fact that the structure of the nucleic acid can be damaged when treated with detergents, in particular alkalis.
Classification of research methods
All molecular biological research methods are divided into three large groups:
1. Amplification (using many enzymes). Hererefers to PCR - polymerase chain reaction, which plays a large role in many of the diagnostic methods.
2. Non-amplifying. This group of methods is directly related to the operation of mixtures of nucleic acids. Examples are 3 blots, in situ hybridization, etc.
3. Methods based on the recognition of a signal from a probe molecule that binds to a specific probe DNA or RNA. An example is the Hybride Capture System (hc2).
Enzymes that can be used in molecular biology research methods
Many molecular diagnostic methods involve the use of a wide range of enzymes. Below are the most commonly used:
1. Restriction enzyme - "cuts" the DNA molecule into the necessary parts.
2. DNA polymerase - synthesizes a double-stranded molecule of deoxyribonucleic acid.
3. Reverse transcriptase (revertase) - used to synthesize DNA on an RNA template.
4. DNA ligase - responsible for the formation of phosphodiester bonds between nucleotides.
5. Exonuclease - removes nucleotides from the terminal sections of the deoxyribonucleic acid molecule.
PCR is the main method of DNA amplification
Polymerase chain reaction (PCR) is actively used in modern molecular biology. This is a method in which a huge number of copies can be obtained from a single DNA molecule (molecules are amplified).
Main functions of PCR:
- diagnosticsdiseases;
- cloning of DNA segments, genes.
The following elements are required to carry out a polymerase chain reaction: the initial DNA molecule, a thermostable DNA polymerase (Taq or Pfu), deoxyribonucleotide phosphates (sources of nitrogenous bases), primers (2 primers per 1 DNA molecule) and the buffer system itself, in which all reactions are possible.
PCR consists of three steps: denaturation, primer annealing and elongation.
1. Denaturation. At a temperature of 94-95 degrees Celsius, the hydrogen bonds between the two strands of DNA are broken, and as a result we get two single-stranded molecules.
2. Primer annealing. At a temperature of 50-60 degrees Celsius, primers are attached to the ends of single-stranded nucleic acid molecules by the type of complementarity.
3. Elongation. At a temperature of 72 degrees, the synthesis of daughter double-stranded molecules of deoxyribonucleic acid occurs.
DNA sequencing
Molecular biological research methods often require knowledge of the nucleotide sequence in a deoxyribonucleic acid molecule. Sequencing is carried out to determine the genetic code. Molecular diagnostics of the future will be based on knowledge gained from human sequencing.
The following types of sequencing are distinguished:
- Maxam-Gilbert sequencing;
- Sanger sequencing;
- pyrosequencing;
- nanoporesequencing.
