Molecular biological research methods play a large role in modern medicine, forensics and biology. Thanks to advances in the study of DNA and RNA, a person is able to study the body’s genome, determine the causative agent of the disease, recognize the desired nucleic acid in a mixture of acids, etc.
Molecular biological research methods. What it is?
Back in the 70-80s, scientists for the first time managed to decipher the human genome. This event gave impetus to the development of genetic engineering and molecular biology. Studying the properties of DNA and RNA has led to the fact that these nucleic acids can now be used to diagnose a disease and study genes.
Obtaining DNA and RNA
Molecular biological methods of diagnosis require the availability of source material: most often these are 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 this must be taken into account when choosing a method for the isolation of nucleic acids in pure form.
1. Obtaining DNA according to Marmur. The method consists in processing 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. DNA isolation by Boom. The main substance used here is guanidine thiocyanate (GuSCN). It promotes the deposition of deoxyribonucleic acid on specialized substrates, from which it can subsequently be collected using a special buffer. However, GuSCN is a PTC inhibitor, and even a small part of it that has fallen into precipitated DNA can affect the course of the polymerase chain reaction, which plays an important role when working with nucleic acids.
3. Precipitation of impurities. The method differs from the previous ones in that it is not the deoxyribonucleic acid molecules themselves that precipitate, but the impurities. To do this, use ion exchangers. The disadvantage is that not all substances can precipitate.
4. Mass screening. This method is used in cases where accurate information about the composition of the DNA molecule is not needed, but some statistical data is needed. This is explained by the fact that the structure of the nucleic acid can be damaged during processing 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). This includes PCR, a polymerase chain reaction that plays a large role in many of the diagnostic methods.
2. Non-amplification. This group of methods is directly related to the work of nucleic acid mixtures. Examples are 3 types of blotting, in situ hybridization, etc.
3. Methods based on the recognition of a signal from a probe molecule that binds to a specific DNA or RNA probe. An example is the Hybride Capture System (hc2) solution hybridization system.
Enzymes that can be used in molecular biological 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 matrix.
4. DNA ligase - is responsible for the formation of phosphodiester bonds between nucleotides.
5. Exonuclease - removes nucleotides from the end sections of a deoxyribonucleic acid molecule.
PCR - the main way to amplify DNA
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 one DNA molecule (to amplify molecules).
The main functions of PCR:
- diagnosis of diseases;
- cloning of DNA sections, genes.
To carry out a polymerase chain reaction, the following elements are necessary: the original DNA molecule, thermostable DNA polymerase (Taq or Pfu), deoxyribonucleotide phosphates (sources of nitrogen bases), primers (2 primers per 1 DNA molecule) and the buffer system itself , in which it is possible to carry out all reactions.
PCR consists of three stages: denaturation, annealing of primers and elongation.
1. Denaturation. At a temperature of 94-95 degrees Celsius breaks the hydrogen bonds between the two DNA chains, and as a result we get two single-stranded molecules.
2. Annealing of primers. At a temperature of 50-60 degrees Celsius, primers are attached at the ends of single-stranded nucleic acid molecules according to 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 performed to determine the genetic code. Molecular diagnostics of the future will be based on the knowledge gained in determining the sequence of a person.
The following types of sequencing are distinguished:
- Maxam-Gilbert sequencing;
- Sanger sequencing;
- pyrosequencing;
- nanopore sequencing.