Applied molecular biology. Molecular biologist. Job Description Molecular Biology Methods

Advances in the study of nucleic acids and protein biosynthesis have led to the creation of a number of methods of great practical importance in medicine, agriculture, and a number of other industries.

After the genetic code and the basic principles of storing and implementing hereditary information were studied, the development of molecular biology came to a standstill, since there were no methods that made it possible to manipulate genes, isolate and change them. The emergence of these methods occurred in the 1970-1980s. This gave a powerful impetus to the development of this field of science, which is still flourishing today. First of all, these methods concern obtaining individual genes and their introduction into cells of other organisms (molecular cloning and transgenesis, PCR), as well as methods for determining the nucleotide sequence in genes (DNA and RNA sequencing). These methods will be discussed in more detail below. We will start with the simplest basic method, electrophoresis, and then move on to more complex methods.

DNA ELECTROPHORESIS

It is the basic method of working with DNA, which is used along with almost all other methods to isolate the desired molecules and analyze the results. Gel electrophoresis is used to separate DNA fragments by length. DNA is an acid, its molecules contain phosphoric acid residues, which split off a proton and acquire a negative charge (Fig. 1).

Therefore, in electric field DNA molecules move towards the anode - a positively charged electrode. This occurs in an electrolyte solution containing charge carrier ions, due to which this solution conducts current. To separate the fragments, a dense gel made of polymers (agarose or polyacrylamide) is used. DNA molecules "entangle" in it the more, the longer they are, and therefore the longest molecules move the slowest, and the shortest - the fastest (Fig. 2). Before or after electrophoresis, the gel is treated with dyes that bind to DNA and fluoresce in ultraviolet light, and a pattern of bands in the gel is obtained (see Fig. 3). To determine the lengths of DNA fragments in a sample, they are compared with a marker, i.e., a set of fragments of standard lengths deposited in parallel on the same gel (Fig. 4).

The most important tools for working with DNA are enzymes that carry out DNA transformations in living cells: DNA polymerases, DNA ligases, and restriction endonucleases, or restriction enzymes. DNA polymerase DNA template synthesis is carried out, which allows DNA to be propagated in a test tube. DNA ligases sew DNA molecules together or heal the gaps in them. Restriction endonucleases, or restrictases, cut DNA molecules according to strictly defined sequences, which allows you to cut out individual fragments from the total mass of DNA. These fragments may in some cases contain individual genes.

restrictases

Sequences recognized by restriction enzymes are symmetrical, and breaks can occur in the middle of such a sequence or with a shift (in the same place in both strands of DNA). Scheme of action different types restrictase is shown in Fig. 1. In the first case, the so-called "blunt" ends are obtained, and in the second - "sticky" ends. In the case of "sticky" ends of the bottom, the chain is shorter than the other, a single-stranded section is formed with a symmetrical sequence that is the same at both ends formed.

The end sequences will be the same when any DNA is cleaved with a given restriction enzyme and can be rejoined because they have complementary sequences. They can be ligated with DNA ligase to form a single molecule. Thus, it is possible to combine fragments of two different DNA and get the so-called recombinant DNA. This approach is used in the method of molecular cloning, which makes it possible to obtain individual genes and introduce them into cells that can form the protein encoded in the gene.

molecular cloning

Molecular cloning uses two DNA molecules - an insert containing the gene of interest, and vector- DNA acting as a carrier. The insert is "sewn" into the vector with the help of enzymes, obtaining a new, recombinant DNA molecule, then this molecule is introduced into host cells, and these cells form colonies on a nutrient medium. A colony is a progeny of one cell, i.e. a clone, all cells of the colony are genetically identical and contain the same recombinant DNA. Hence the term "molecular cloning", that is, obtaining a clone of cells containing a DNA fragment of interest to us. After the colonies containing the insert of interest to us have been obtained, we can various methods to characterize this insertion, for example, to determine its exact sequence. The cells can also produce the protein encoded by the insert if it contains a functional gene.

When a recombinant molecule is introduced into cells, the genetic transformation of these cells occurs. Transformation- the process of absorption by a cell of an organism of a free DNA molecule from the environment and its integration into the genome, which leads to the appearance in such a cell of new heritable traits for it, characteristic of the organism-donor of DNA. For example, if the inserted molecule contains a gene for resistance to the antibiotic ampicillin, then the transformed bacteria will grow in its presence. Before transformation, ampicillin caused their death, that is, a new sign appears in the transformed cells.

VECTORS

A vector must have a number of properties:

First, it is a relatively small DNA molecule to be easily manipulated.

Secondly, in order for DNA to be preserved and reproduced in a cell, it must contain a certain sequence that ensures its replication (the origin of replication, or origin of replication).

Thirdly, it must contain marker gene, which ensures the selection of only those cells into which the vector has entered. Usually these are antibiotic resistance genes - then in the presence of an antibiotic, all cells that do not contain the vector die.

Gene cloning is most often carried out in bacterial cells, as they are easy to cultivate and multiply rapidly. In a bacterial cell, there is usually one large circular DNA molecule, several million base pairs long, containing all the genes necessary for bacteria - the bacterial chromosome. In addition to it, in some bacteria there are small (several thousand base pairs) circular DNA, called plasmids(Fig. 2). They, like the main DNA, contain a nucleotide sequence that provides the ability of DNA to replicate (ori). Plasmids replicate independently of the main (chromosomal) DNA, therefore they are present in the cell in a large number of copies. Many of these plasmids carry antibiotic resistance genes, which makes it possible to distinguish cells carrying the plasmid from normal cells. More commonly, plasmids carrying two genes conferring resistance to two antibiotics, such as tetracycline and amycilin, are used. There are simple methods for isolating such plasmid DNA free from the DNA of the main chromosome of the bacterium.

THE SIGNIFICANCE OF TRANSGENESIS

The transfer of genes from one organism to another is called transgenesis, and such modified organisms - transgenic. The method of gene transfer into microbial cells is used to obtain recombinant protein preparations for medicine, in particular, human proteins that do not cause immune rejection - interferons, insulin and other protein hormones, cell growth factors, as well as proteins for the production of vaccines. In more complex cases, when protein modification is carried out correctly only in eukaryotic cells, transgenic cell cultures or transgenic animals are used, in particular, livestock (primarily goats), which secrete the necessary proteins into milk, or proteins are isolated from their blood. This is how antibodies, blood clotting factors and other proteins are obtained. By the method of transgenesis, cultivated plants are obtained that are resistant to herbicides and pests and have other useful properties. Using transgenic microorganisms to purify wastewater and fight pollution, there are even transgenic microbes that can break down oil. In addition, transgenic technologies are indispensable in scientific research - the development of biology today is unthinkable without the routine use of gene modification and transfer methods.

molecular cloning technology

inserts

To obtain an individual gene from any organism, all chromosomal DNA is isolated from it and cleaved with one or two restriction enzymes. Enzymes are selected so that they do not cut the gene of interest to us, but make breaks along its edges, and in plasmid DNA make one break in one of the resistance genes, for example, to ampicillin.

The molecular cloning process includes the following steps:

Cut and stitch - construction of a single recombinant molecule from an insert and a vector.

Transformation is the introduction of a recombinant molecule into cells.

Selection - selection of cells that received a vector with an insert.

cutting and stitching

Plasmid DNA is treated with the same restriction enzymes, and it turns into a linear molecule if such a restriction enzyme is selected that introduces 1 break into the plasmid. As a result, the same sticky ends appear at the ends of all the resulting DNA fragments. As the temperature is lowered, these ends join randomly and are ligated with DNA ligase (see Fig. 3).

A mixture of circular DNAs of different composition is obtained: some of them will contain a certain DNA sequence of chromosomal DNA connected to bacterial DNA, others will contain fragments of chromosomal DNA joined together, and still others will contain a reduced circular plasmid or its dimer (Fig. 4).

transformation

Next, this mixture is carried out genetic transformation bacteria that do not contain plasmids. Transformation- the process of absorption by a cell of an organism of a free DNA molecule from the environment and its integration into the genome, which leads to the appearance in such a cell of new heritable traits for it, characteristic of the organism-donor of DNA. Only one plasmid can enter and multiply in each cell. Such cells are placed on a solid nutrient medium containing the antibiotic tetracycline. Cells that did not get the plasmid will not grow on this medium, and the cells carrying the plasmid form colonies, each of which contains the descendants of only one cell, i.e. all cells in a colony carry the same plasmid (see Fig. 5).

Selection

Next, the task is to isolate only the cells into which the vector with the insert has entered, and to distinguish them from cells carrying only the vector without the insert or not carrying the vector at all. This process of selecting the right cells is called selection. For this, apply selective markers- usually antibiotic resistance genes in the vector, and selective media containing antibiotics or other selective substances.

In the example we are considering, cells from colonies grown in the presence of ampicillin are subcultured on two media: the first contains ampicillin, and the second contains tetracycline. Colonies containing only the plasmid will grow on both media, while colonies containing inserted chromosomal DNA in the plasmids will not grow on the medium with tetracycline (Fig. 5). Among them, those that contain the gene of interest to us are selected by special methods, grown in sufficient quantities, and plasmid DNA is isolated. From it, using the same restrictases that were used to obtain recombinant DNA, the individual gene of interest is cut out. The DNA of this gene can be used to determine the sequence of nucleotides, introduce into any organism to obtain new properties or synthesize the desired protein. This method of gene isolation is called molecular cloning.

FLUORESCENT PROTEINS

It is very convenient to use fluorescent proteins as marker genes in studies of eukaryotic organisms. The gene for the first fluorescent protein, green fluorescent protein (GFP) was isolated from the jellyfish Aqeuorea victoria and introduced into various model organisms (see Fig. 6) In 2008, O. Shimomura, M. Chalfi and R. Tsien received the Nobel Prize for the discovery and application of this protein.

Then the genes for other fluorescent proteins - red, blue, yellow - were isolated. These genes have been artificially modified to produce proteins with desired properties. The diversity of fluorescent proteins is shown in fig. 7, which shows a petri dish with bacteria containing genes for various fluorescent proteins.

application of fluorescent proteins

The fluorescent protein gene can be fused with the gene of any other protein, then during translation a single protein will be formed - a translational fusion protein, or fusion(fusion protein), which fluoresces. Thus, it is possible to study, for example, the localization (location) of any proteins of interest in the cell, their movement. Using the expression of fluorescent proteins only in certain types of cells, it is possible to mark cells of these types in a multicellular organism (see Fig. 8 - mouse brain, in which individual neurons have different colors due to a certain combination of fluorescent protein genes). Fluorescent proteins are an indispensable tool in modern molecular biology.

PCR

Another method for obtaining genes is called polymerase chain reaction (PCR). It is based on the ability of DNA polymerases to complete the second strand of DNA along the complementary strand, as occurs in cells during DNA replication.

The origins of replication in this method are given by two small pieces of DNA called seeds, or primers. These primers are complementary to the ends of the gene of interest on two strands of DNA. First, the chromosomal DNA from which the gene is to be isolated is mixed with seeds and heated to 99 ° C. This leads to the breaking of hydrogen bonds and the divergence of DNA strands. After that, the temperature is lowered to 50-70 about C (depending on the length and sequence of seeds). Under these conditions, the primers are attached to complementary regions of chromosomal DNA, forming a regular double helix (see Fig. 9). After that, a mixture of all four nucleotides needed for DNA synthesis and DNA polymerase are added. The enzyme elongates the primers by building double-stranded DNA from the point of attachment of the primers, i.e. from the ends of a gene to the end of a single-stranded chromosome molecule.

If the mixture is now heated again, the chromosomal and newly synthesized chains will disperse. After cooling, seeds will again join them, which are taken in large excess (see Fig. 10).

On the newly synthesized chains, they will join not to the end from which the first synthesis began, but to the opposite one, since the DNA chains are antiparallel. Therefore, in the second cycle of synthesis, only the sequence corresponding to the gene will be completed on such chains (see Fig. 11).

This method uses DNA polymerase from thermophilic bacteria that can withstand boiling and operates at temperatures of 70-80 ° C, it does not need to be added every time, but it is enough to add it at the beginning of the experiment. By repeating the heating and cooling procedures in the same sequence, we can double the number of sequences in each cycle, bounded at both ends by the introduced seeds (see Fig. 12).

After about 25 such cycles, the number of copies of the gene will increase by more than a million times. Such quantities can be easily separated from the chromosomal DNA introduced into the test tube and used for various purposes.

DNA sequencing

Another important achievement is the development of methods for determining the sequence of nucleotides in DNA - DNA sequencing(from English sequence - sequence). To do this, it is necessary to obtain genes pure from other DNA using one of the described methods. Then the DNA chains are separated by heating and a primer labeled with radioactive phosphorus or a fluorescent label is added to them. Please note that one seed is taken, complementary to one chain. Then DNA polymerase and a mixture of 4 nucleotides are added. Such a mixture is divided into 4 parts and one of the nucleotides is added to each, modified so that it does not contain a hydroxyl group on the third atom of deoxyribose. If such a nucleotide is included in the synthesized DNA chain, then its elongation will not be able to continue, because polymerase will have nowhere to attach the next nucleotide. Therefore, DNA synthesis after the inclusion of such a nucleotide is interrupted. These nucleotides, called dideoxynucleotides, are added much less than usual, so chain termination occurs only occasionally and in each chain in different places. The result is a mixture of chains different lengths, at the end of each of them is the same nucleotide. Thus, the chain length corresponds to the nucleotide number in the studied sequence, for example, if we had an adenyl dideoxynucleotide, and the resulting chains were 2, 7 and 12 nucleotides long, then adenine was in the second, seventh and twelfth positions in the gene. The resulting mixture of chains can be easily separated by size using electrophoresis, and the synthesized chains can be identified by radioactivity on X-ray film (see Fig. 10).

It turns out the picture shown at the bottom of the picture, called radioautograph. Moving along it from bottom to top and reading the letter above the columns of each zone, we will get the nucleotide sequence shown in the figure to the right of the autograph. It turned out that synthesis is stopped not only by dideoxynucleotides, but also by nucleotides in which some chemical group, for example, a fluorescent dye, is attached to the third position of the sugar. If each nucleotide is labeled with its own dye, then the zones obtained by separating the synthesized chains will glow with a different light. This makes it possible to carry out the reaction in one test tube simultaneously for all nucleotides and, by separating the resulting chains by length, to identify the nucleotides by color (see Fig. 11).

Such methods made it possible to determine the sequences not only of individual genes, but also to read entire genomes. Even faster methods for determining nucleotide sequences in genes have now been developed. If the first human genome was deciphered by a large international consortium using the first given method in 12 years, the second, using the second, in three years, now this can be done in a month. This allows you to predict a person's predisposition to many diseases and take measures in advance to avoid them.

Comic book for the contest "bio/mol/text": Today, the molecular biologist Test Tube will guide you through the world of amazing science - molecular biology! We will start with a historical excursion through the stages of its development, we will describe the main discoveries and experiments since 1933. And we will also clearly describe the main methods of molecular biology, which made it possible to manipulate genes, change and isolate them. The emergence of these methods served as a strong impetus to the development of molecular biology. And let's also remember the role of biotechnology and touch on one of the most popular topics in this area - genome editing using CRISPR/Cas systems.

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1. Introduction. Essence of molecular biology

It studies the basics of the vital activity of organisms at the level of macromolecules. The goal of molecular biology is to establish the role and mechanisms of functioning of these macromolecules on the basis of knowledge about their structures and properties.

Historically, molecular biology was formed during the development of areas of biochemistry that study nucleic acids and proteins. While biochemistry is the study of metabolism, chemical composition living cells, organisms and the chemical processes carried out in them, molecular biology focuses on the study of the mechanisms of transmission, reproduction and storage of genetic information.

And the object of study of molecular biology is the nucleic acids themselves - deoxyribonucleic (DNA), ribonucleic (RNA) - and proteins, as well as their macromolecular complexes - chromosomes, ribosomes, multienzyme systems that provide the biosynthesis of proteins and nucleic acids. Molecular biology also borders on the objects of research and partially coincides with molecular genetics, virology, biochemistry and a number of other related biological sciences.

2. Historical excursion through the stages of development of molecular biology

As a separate area of biochemistry, molecular biology began to develop in the 30s of the last century. Even then, it became necessary to understand the phenomenon of life at the molecular level in order to study the processes of transmission and storage of genetic information. Just at that time, the task of molecular biology was established in the study of the properties, structure and interaction of proteins and nucleic acids.

The term "molecular biology" was first used in 1933 year William Astbury during the study of fibrillar proteins (collagen, blood fibrin, contractile muscle proteins). Astbury studied the relationship between the molecular structure and the biological, physical characteristics of these proteins. At the beginning of the emergence of molecular biology, RNA was considered to be a component only of plants and fungi, and DNA - only animals. And in 1935 The discovery of pea DNA by Andrei Belozersky led to the establishment of the fact that DNA is contained in every living cell.

IN 1940 A colossal achievement was the establishment by George Beadle and Edward Tatham of a causal relationship between genes and proteins. The scientists' hypothesis "One gene - one enzyme" formed the basis for the concept that the specific structure of a protein is regulated by genes. It is believed that genetic information is encoded by a special sequence of nucleotides in DNA that regulates the primary structure of proteins. Later it was proved that many proteins have a quaternary structure. Various peptide chains take part in the formation of such structures. Based on this, the provision on the relationship between a gene and an enzyme has been somewhat transformed, and now it sounds like "One gene - one polypeptide."

IN 1944 In 1999, the American biologist Oswald Avery and his colleagues (Colin McLeod and McLean McCarthy) proved that the substance that causes the transformation of bacteria is DNA, not proteins. The experiment served as proof of the role of DNA in the transmission of hereditary information, crossing out outdated knowledge about the protein nature of genes.

In the early 1950s, Frederick Sanger showed that a protein chain is a unique sequence of amino acid residues. IN 1951 And 1952 years, the scientist determined the complete sequence of two polypeptide chains - bovine insulin IN(30 amino acid residues) and A(21 amino acid residues), respectively.

Around the same time, in 1951–1953 Erwin Chargaff formulated the rules for the ratio of nitrogenous bases in DNA. According to the rule, regardless of the species differences of living organisms in their DNA, the amount of adenine (A) is equal to the amount of thymine (T), and the amount of guanine (G) is equal to the amount of cytosine (C).

IN 1953 proved the genetic role of DNA. James Watson and Francis Crick, based on the X-ray of DNA obtained by Rosalind Franklin and Maurice Wilkins, established the spatial structure of DNA and put forward a later confirmed assumption about the mechanism of its replication (doubling), which underlies heredity.

1958 year - the formation of the central dogma of molecular biology by Francis Crick: the transfer of genetic information goes in the direction of DNA → RNA → protein.

The essence of the dogma is that in cells there is a certain directed flow of information from DNA, which, in turn, is the original genetic text, consisting of four letters: A, T, G and C. It is written in the DNA double helix in the form sequences of these letters - nucleotides.

This text is being transcribed. And the process is called transcription. During this process, RNA is synthesized, which is identical to the genetic text, but with a difference: in RNA, instead of T, there is U (uracil).

This RNA is called messenger RNA (mRNA), or matrix (mRNA). Broadcast mRNA is carried out using the genetic code in the form of triplet sequences of nucleotides. During this process, the text of DNA and RNA nucleic acids is translated from a four-letter text into a twenty-letter text of amino acids.

There are only twenty natural amino acids, and there are four letters in the text of nucleic acids. Because of this, there is a translation from the four-letter alphabet to the twenty-letter alphabet through the genetic code, in which each three nucleotides corresponds to an amino acid. So you can make whole 64 three-letter combinations from four letters, moreover, there are 20 amino acids. From this it follows that the genetic code must necessarily have the property of degeneracy. However, at that time the genetic code was not known, besides, it had not even begun to be deciphered, but Crick had already formulated his central dogma.

Nevertheless, there was a certainty that the code must exist. By that time, it had been proved that this code had a triplet character. This means that specifically three letters in nucleic acids ( codons) correspond to any amino acid. There are 64 of these codons, they code for 20 amino acids. This means that each amino acid corresponds to several codons at once.

Thus, we can conclude that the central dogma is a postulate that says that a directed flow of information occurs in the cell: DNA → RNA → protein. Crick emphasized the main content of the central dogma: a reverse flow of information cannot occur, a protein is not capable of changing genetic information.

This is the main meaning of the central dogma: a protein is not able to change and transform information into DNA (or RNA), the flow always goes only in one direction.

Some time after this, a new enzyme was discovered, which was not known at the time of the formulation of the central dogma, - reverse transcriptase that synthesizes DNA from RNA. The enzyme was discovered in viruses, in which the genetic information is encoded in RNA, not DNA. Such viruses are called retroviruses. They have a viral capsule with RNA enclosed in it and a special enzyme. The enzyme is a reverse transcriptase that synthesizes DNA according to the template of this viral RNA, and this DNA then serves as the genetic material for the further development of the virus in the cell.

Of course, this discovery caused great shock and much controversy among molecular biologists, since it was believed that, based on central dogma, this could not be. However, Crick immediately explained that he never said it was impossible. He only said that there can never be a flow of information from protein to nucleic acids, and already inside nucleic acids any kind of processes are quite possible: the synthesis of DNA on DNA, DNA on RNA, RNA on DNA and RNA on RNA.

After the formulation of the central dogma, a number of questions still remained: how does the alphabet of four nucleotides that make up DNA (or RNA) encode the 20-letter alphabet of amino acids that make up proteins? What is the essence of the genetic code?

The first ideas about the existence of the genetic code were formulated by Alexander Downes ( 1952 d.) and Georgy Gamov ( 1954 G.). Scientists have shown that the sequence of nucleotides must include at least three links. Later it was proved that such a sequence consists of three nucleotides, called codon (triplet). However, the question of which nucleotides are responsible for incorporating which amino acid into a protein molecule remained open until 1961.

And in 1961 Marshall Nirenberg, along with Heinrich Mattei, used the system to broadcast in vitro. An oligonucleotide was used as a template. It contained only uracil residues, and the peptide synthesized from it included only the amino acid phenylalanine. Thus, the meaning of the codon was first established: the codon UUU codes for phenylalanine. Later, Har Qur'an found that the nucleotide sequence UCUCUCUCUCUC encodes a set of amino acids serine-leucine-serine-leucine. By and large, thanks to the works of Nirenberg and the Koran, to 1965 year, the genetic code was completely unraveled. It turned out that each triplet encodes a specific amino acid. And the order of the codons determines the order of the amino acids in the protein.

The main principles of the functioning of proteins and nucleic acids were formulated by the beginning of the 70s. It was found that the synthesis of proteins and nucleic acids is carried out according to the matrix mechanism. The template molecule carries encoded information about the sequence of amino acids or nucleotides. During replication or transcription, the template is DNA, and during translation and reverse transcription, it is mRNA.

Thus, the prerequisites for the formation of areas of molecular biology, including genetic engineering, were created. And in 1972, Paul Berg and colleagues developed the technology of molecular cloning. Scientists have obtained the first recombinant DNA in vitro. These outstanding discoveries formed the basis of a new direction in molecular biology, and 1972 the year has since been considered the birth date of genetic engineering.

3. Methods of molecular biology

Enormous advances in the study of nucleic acids, the structure of DNA and protein biosynthesis have led to the creation of a number of methods of great importance in medicine, agriculture and science in general.

After studying the genetic code and the basic principles of storage, transmission and implementation of hereditary information, special methods became necessary for the further development of molecular biology. These methods would allow genes to be manipulated, altered and isolated.

The emergence of such methods occurred in the 1970s and 1980s. This gave a huge impetus to the development of molecular biology. First of all, these methods are directly related to the production of genes and their introduction into the cells of other organisms, as well as the possibility of determining the nucleotide sequence in genes.

3.1. DNA electrophoresis

DNA electrophoresis is the basic method of working with DNA. DNA electrophoresis is used along with almost all other methods to isolate the desired molecules and further analyze the results. The gel electrophoresis method itself is used to separate DNA fragments by length.

Before or after electrophoresis, the gel is treated with dyes that can bind to DNA. The dyes fluoresce in ultraviolet light, resulting in a pattern of bands in the gel. To determine the length of DNA fragments, they can be compared with markers- sets of fragments of standard lengths, which are applied to the same gel.

Fluorescent proteins

When studying eukaryotic organisms, it is convenient to use fluorescent proteins as marker genes. The gene for the first green fluorescent protein ( green fluorescent protein, GFP) isolated from jellyfish Aqeuorea victoria and then introduced into various organisms. After that, genes for fluorescent proteins of other colors were isolated: blue, yellow, red. To obtain proteins with properties of interest, such genes have been artificially modified.

In general, the most important tools for working with the DNA molecule are enzymes that carry out a number of DNA transformations in cells: DNA polymerase, DNA ligases And restrictases (restriction endonucleases).

transgenesis

transgenesis It is called the transfer of genes from one organism to another. Such organisms are called transgenic.

Recombinant protein preparations are just obtained by transferring genes into microorganism cells. Most of these proteins are interferons, insulin, some protein hormones, as well as proteins for the production of a number of vaccines.

In other cases, cell cultures of eukaryotes or transgenic animals, mostly livestock, are used, which secrete the necessary proteins into milk. In this way, antibodies, blood clotting factors and other proteins are obtained. The transgenesis method is used to obtain crops resistant to pests and herbicides, and wastewater is treated with the help of transgenic microorganisms.

In addition to all of the above, transgenic technologies are indispensable in scientific research, because the development of biology is faster with the use of gene modification and transfer methods.

Restrictases

The sequences recognized by restriction enzymes are symmetrical, so any kind of breaks can occur either in the middle of such a sequence, or with a shift in one or both strands of the DNA molecule.

When splitting any DNA with a restriction enzyme, the sequences at the ends of the fragments will be the same. They will be able to connect again because they have complementary sites.

You can get a single molecule by stitching these sequences using DNA ligases. Due to this, it is possible to combine fragments of two different DNA and obtain recombinant DNA.

3.2. PCR

The method is based on the ability of DNA polymerases to complete the second strand of DNA along the complementary strand in the same way as in the process of DNA replication in a cell.

3.3. DNA sequencing

The rapid development of the sequencing method makes it possible to effectively determine the characteristics of the organism under study at the level of its genome. The main advantage of such genomic and post-genomic technologies is the increase in research and study opportunities. genetic nature human diseases, in order to pre-take necessary measures and avoid illness.

Through large-scale research, it is possible to obtain the necessary data on the various genetic characteristics of different groups of people, thereby developing the methods of medicine. Because of this, the identification of a genetic predisposition to various diseases is very popular today.

Similar methods are widely applicable practically all over the world, including in Russia. Due to scientific progress, such methods are being introduced into medical research and medical practice generally.

4. Biotechnology

Biotechnology- a discipline that studies the possibilities of using living organisms or their systems to solve technological problems, as well as creating living organisms with the desired properties through genetic engineering. Biotechnology applies the methods of chemistry, microbiology, biochemistry and, of course, molecular biology.

The main directions of development of biotechnology (the principles of biotechnological processes are being introduced into the production of all industries):

Creation and production of new types of food and animal feed.
Obtaining and studying new strains of microorganisms.
Breeding of new varieties of plants, as well as the creation of means for protecting plants from diseases and pests.
Application of biotechnology methods for the needs of ecology. Such methods of biotechnology are used for recycling waste disposal, cleaning Wastewater, exhaust air and sanitation of soils.
Production of vitamins, hormones, enzymes, serums for the needs of medicine. Biotechnologists are developing improved medications previously considered incurable.

A major achievement in biotechnology is genetic engineering.

Genetic Engineering- a set of technologies and methods for obtaining recombinant RNA and DNA molecules, isolating individual genes from cells, manipulating genes and introducing them into other organisms (bacteria, yeast, mammals). Such organisms are able to produce final products with the desired, modified properties.

Genetic engineering methods are aimed at constructing new, previously non-existing combinations of genes in nature.

Speaking about the achievements of genetic engineering, it is impossible not to touch on the topic of cloning. Cloning is one of the methods of biotechnology used to obtain identical offspring of different organisms through asexual reproduction.

In other words, cloning can be thought of as the process of creating genetically identical copies of an organism or cell. And cloned organisms are similar or completely identical not only in external features, but also in genetic content.

The notorious sheep Dolly in 1966 became the first cloned mammal. It was obtained by transplanting the nucleus of a somatic cell into the cytoplasm of the egg. Dolly was a genetic copy of the nucleus donor sheep. Under natural conditions, an individual is formed from one fertilized egg, having received half of the genetic material from two parents. However, during cloning, the genetic material was taken from the cell of one individual. First, the nucleus, which contains the DNA itself, was removed from the zygote. Then they removed the nucleus from the adult sheep cell and implanted it into that zygote without the nucleus, and then it was transplanted into the uterus of an adult and allowed to grow and develop.

However, not all cloning attempts have been successful. In parallel with Dolly's cloning, a DNA replacement experiment was carried out on 273 other eggs. But only in one case could a living adult animal fully develop and grow. After Dolly, scientists tried to clone other types of mammals.

One of the types of genetic engineering is genome editing.

The CRISPR/Cas tool is based on an element of the immune defense system of bacteria, which scientists have adapted to introduce any changes in the DNA of animals or plants.

CRISPR/Cas is one of the biotechnological methods for manipulating individual genes in cells. There are many applications for this technology. CRISPR/Cas allows researchers to figure out the function of different genes. To do this, you just need to cut out the gene under study from the DNA and study which functions of the body were affected.

Some practical applications of the system:

Agriculture. Through CRISPR/Cas systems, crops can be improved. Namely, to make them more tasty and nutritious, as well as resistant to heat. It is possible to endow plants with other properties: for example, cut out an allergen gene from nuts (peanuts or hazelnuts).
Medicine, hereditary diseases. Scientists have a goal to use CRISPR/Cas to remove mutations from the human genome that can cause diseases, such as sickle cell anemia, etc. In theory, CRISPR/Cas can stop the development of HIV.
Gene drive. CRISPR/Cas can change not only the genome of an individual animal or plant, but also the gene pool of a species. This concept is known as "gene drive". Every living organism passes on half of its genes to its offspring. But using CRISPR/Cas can increase the chance of gene transfer by up to 100%. This is important in order for the desired trait to spread faster throughout the population.

Swiss scientists have significantly improved and modernized the CRISPR/Cas genome editing method, thereby expanding its capabilities. However, scientists could only modify one gene at a time using the CRISPR/Cas system. But now researchers at ETH Zurich have developed a method that can simultaneously modify 25 genes in a cell.

For the latest technique, experts used the Cas12a enzyme. Geneticists have successfully cloned monkeys for the first time in history. "Popular Mechanics";

Nikolenko S. (2012). Genomics: Problem Statement and Sequencing Methods. "Post-science".

Molecular biology has experienced a period of rapid development of its own research methods, which now differs from biochemistry. These include, in particular, methods of genetic engineering, cloning, artificial expression, and gene knockout. Since DNA is the material carrier of genetic information, molecular biology has become much closer to genetics, and molecular genetics was formed at the junction, which is both a section of genetics and molecular biology. Just as molecular biology makes extensive use of viruses as a research tool, virology uses the methods of molecular biology to solve its problems. Computer technology is involved in the analysis of genetic information, in connection with which new areas of molecular genetics have appeared, which are sometimes considered special disciplines: bioinformatics, genomics and proteomics.

History of development

This seminal discovery was prepared by a long phase of research into the genetics and biochemistry of viruses and bacteria.

In 1928, Frederick Griffith first showed that an extract of heat-killed pathogenic bacteria could transfer the trait of pathogenicity to benign bacteria. The study of bacterial transformation further led to the purification of the disease agent, which, contrary to expectations, turned out to be not a protein, but a nucleic acid. The nucleic acid itself is not dangerous, it only carries the genes that determine the pathogenicity and other properties of the microorganism.

In the 50s of the XX century, it was shown that bacteria have a primitive sexual process, they are able to exchange extrachromosomal DNA, plasmids. The discovery of plasmids, as well as transformations, formed the basis of the plasmid technology common in molecular biology. Another important discovery for the methodology was the discovery at the beginning of the 20th century of bacterial viruses, bacteriophages. Phages can also transfer genetic material from one bacterial cell to another. Infection of bacteria by phages leads to a change in the composition of bacterial RNA. If, without phages, the composition of RNA is similar to the composition of bacterial DNA, then after infection, RNA becomes more similar to bacteriophage DNA. Thus, it was found that the structure of RNA is determined by the structure of DNA. In turn, the rate of protein synthesis in cells depends on the amount of RNA-protein complexes. This is how it was formulated central dogma of molecular biology: DNA ↔ RNA → protein.

The further development of molecular biology was accompanied by both the development of its methodology, in particular, the invention of a method for determining the nucleotide sequence of DNA (W. Gilbert and F. Sanger, Nobel Prize in Chemistry in 1980), and new discoveries in the field of research into the structure and functioning of genes (see. History of genetics). By the beginning of the 21st century, data were obtained on the primary structure of all human DNA and a number of other organisms, the most important for medicine, Agriculture and scientific research, which led to the emergence of several new areas in biology: genomics, bioinformatics, etc.

Literature

Singer M., Berg P. Genes and genomes. - Moscow, 1998.
Stent G., Kalindar R. Molecular genetics. - Moscow, 1981.
Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning. - 1989.
Patrushev L.I. Expression of genes. - M.: Nauka, 2000. - 000 p., ill. ISBN 5-02-001890-2

Links

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See what "Molecular Biology" is in other dictionaries:

MOLECULAR BIOLOGY- studies the basics. properties and manifestations of life at the molecular level. The most important directions in M. b. are studies of the structural and functional organization of the genetic apparatus of cells and the mechanism for the implementation of hereditary information ... ... Biological encyclopedic dictionary

MOLECULAR BIOLOGY- explores the basic properties and manifestations of life at the molecular level. Finds out how and to what extent the growth and development of organisms, the storage and transmission of hereditary information, the conversion of energy in living cells, and other phenomena are due to ... Big Encyclopedic Dictionary

MOLECULAR BIOLOGY Modern Encyclopedia

MOLECULAR BIOLOGY- MOLECULAR BIOLOGY, the biological study of the structure and function of the MOLECULES that make up living organisms. The main areas of study are physical and Chemical properties proteins and NUCLEIC ACIDS such as DNA. see also… … Scientific and technical encyclopedic dictionary

molecular biology- a section of biol., which explores the basic properties and manifestations of life at the molecular level. Finds out how and to what extent the growth and development of organisms, the storage and transmission of hereditary information, the conversion of energy in living cells and ... ... Dictionary of microbiology

molecular biology- — Topics of biotechnology EN molecular biology … Technical Translator's Handbook

Molecular biology- MOLECULAR BIOLOGY, explores the basic properties and manifestations of life at the molecular level. Finds out how and to what extent the growth and development of organisms, the storage and transmission of hereditary information, the conversion of energy in living cells and ... ... Illustrated Encyclopedic Dictionary

Molecular biology- a science that sets as its task the knowledge of the nature of life phenomena by studying biological objects and systems at a level approaching the molecular level, and in some cases reaching this limit. The end goal of this is…… Great Soviet Encyclopedia

MOLECULAR BIOLOGY- studies the phenomena of life at the level of macromolecules (ch. arr. proteins and nucleic acids) in cell-free structures (ribosomes, etc.), in viruses, and also in cells. M.'s purpose. establishing the role and mechanism of functioning of these macromolecules based on ... ... Chemical Encyclopedia

molecular biology- explores the basic properties and manifestations of life at the molecular level. Finds out how and to what extent the growth and development of organisms, the storage and transmission of hereditary information, the conversion of energy in living cells and other phenomena ... ... encyclopedic Dictionary

Books

Molecular biology of the cell. Problem Book, J. Wilson, T. Hunt. The book of American authors is an appendix to the 2nd edition of the textbook `Molecular Biology of the Cell` by B. Alberts, D. Bray, J. Lewis and others. Contains questions and tasks, the purpose of which is to deepen ...

Molecular biology, a science that sets as its task the knowledge of the nature of life phenomena by studying biological objects and systems at a level approaching the molecular level, and in some cases reaching this limit. The ultimate goal in this case is to clarify how and to what extent the characteristic manifestations of life, such as heredity, reproduction of one's own kind, protein biosynthesis, excitability, growth and development, storage and transmission of information, energy transformations, mobility, etc. , are due to the structure, properties and interaction of molecules of biologically important substances, primarily the two main classes of high-molecular biopolymers - proteins and nucleic acids. A distinctive feature of M. b. - the study of the phenomena of life on inanimate objects or those that are characterized by the most primitive manifestations of life. These are biological formations from the cellular level and below: subcellular organelles, such as isolated cell nuclei, mitochondria, ribosomes, chromosomes, cell membranes; further - systems that stand on the border of animate and inanimate nature - viruses, including bacteriophages, and ending with molecules critical components living matter - nucleic acids and proteins.

The foundation on which M. developed. was laid by such sciences as genetics, biochemistry, physiology of elementary processes, etc. According to the origins of its development, M. b. is inextricably linked to molecular genetics, which continues to be an important part of

A distinctive feature of M. b. is its three-dimensionality. The essence of M. b. M. Perutz sees it in interpreting biological functions in terms of molecular structure. M. b. aims to get answers to the question "how", knowing the essence of the role and participation of the entire structure of the molecule, and to the questions "why" and "why", having found out, on the one hand, the relationship between the properties of the molecule (again, primarily proteins and nucleic acids) and the functions it performs and, on the other hand, the role of such individual functions in the overall complex of manifestations of vital activity.

The most important achievements of molecular biology. Here is a far from complete list of these achievements: disclosure of the structure and mechanism of the biological function of DNA, all types of RNA and ribosomes, disclosure of the genetic code; discovery of reverse transcription, i.e., DNA synthesis on an RNA template; study of the mechanisms of functioning of respiratory pigments; discovery of the three-dimensional structure and its functional role in the action of enzymes, the principle of matrix synthesis and the mechanisms of protein biosynthesis; disclosure of the structure of viruses and the mechanisms of their replication, the primary and, in part, the spatial structure of antibodies; isolation of individual genes, chemical and then biological (enzymatic) gene synthesis, including human, outside the cell (in vitro); transfer of genes from one organism to another, including into human cells; the rapidly progressing deciphering of the chemical structure of an increasing number of individual proteins, mainly enzymes, as well as nucleic acids; discovery of the phenomena of "self-assembly" of some biological objects of ever-increasing complexity, starting from nucleic acid molecules and moving on to multicomponent enzymes, viruses, ribosomes, etc.; elucidation of allosteric and other basic principles of regulation of biological functions and processes.

Problems of molecular biology. Along with the specified important tasks M. would. (knowledge of the laws of "recognition", self-assembly and integration) the actual direction of scientific search for the near future is the development of methods that allow deciphering the structure, and then the three-dimensional, spatial organization of high-molecular nucleic acids. All the most important methods, the use of which ensured the emergence and success of M. b., were proposed and developed by physicists (ultracentrifugation, X-ray diffraction analysis, electron microscopy, nuclear magnetic resonance, etc.). Almost all new physical experimental approaches (for example, the use of computers, synchrotron, or bremsstrahlung, radiation, laser technology, and others) open up new possibilities for an in-depth study of the problems of M. b. Among the most important tasks of a practical nature, the answer to which is expected from M. b., in the first place is the problem of the molecular basis of malignant growth, then - ways to prevent, and perhaps overcome hereditary diseases - "molecular diseases". Of great importance will be the elucidation of the molecular basis of biological catalysis, ie, the action of enzymes. Among the most important modern trends M. b. should include the desire to decipher the molecular mechanisms of action of hormones, toxic and medicinal substances, as well as to find out the details of the molecular structure and functioning of such cellular structures as biological membranes involved in the regulation of the processes of penetration and transport of substances. More distant goals M. b. - knowledge of the nature of nervous processes, mechanisms of memory, etc. One of the important emerging sections of M. b. - so-called. genetic engineering, which sets as its task the purposeful operation of the genetic apparatus (genome) of living organisms, starting with microbes and lower (single-celled) and ending with humans (in the latter case, primarily for the purpose of radical treatment of hereditary diseases and correction of genetic defects).

The most important directions of the MB:

- Molecular genetics - the study of the structural and functional organization of the genetic apparatus of the cell and the mechanism for the implementation of hereditary information

– Molecular virology – the study of the molecular mechanisms of the interaction of viruses with cells

– Molecular immunology – the study of patterns of immune reactions of the body

– Molecular biology of development – the study of the appearance of cell diversity in the course of individual development of organisms and specialization of cells

Main objects of research: Viruses (including bacteriophages), Cells and subcellular structures, Macromolecules, Multicellular organisms.