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Molecular biologists study molecular processes as the basis of all life processes. Based on the results obtained, they develop concepts for the use of biochemical processes, for example, in medical research and diagnostics or in biotechnology. In addition, they may be involved in pharmaceutical product manufacturing, product development, quality assurance, or pharmaceutical consulting.
Molecular biologists can work in different fields. For example, they relate to the use of research results for production in areas such as genetic engineering, protein chemistry or pharmacology (drug discovery). In the chemical and pharmaceutical industries, they facilitate the introduction of newly developed products from research into production, product marketing and user consulting.
In scientific research, molecular biologists study the chemical and physical properties of organic compounds, as well as chemical processes (in the field of cellular metabolism) in living organisms and publish the results of the research. In higher education institutions, they teach students, prepare for lectures and seminars, check written work, and take exams. Independent scientific activity is possible only after obtaining a master's and doctor's degrees.
Molecular biologists find jobs such as
The salary received by Molecular Biologists in Germany is
(according to various statistical offices and employment services in Germany)
Molecular biologists study molecular processes as the basis of all life processes. Based on the results obtained, they develop concepts for the use of biochemical processes, for example, in medical research and diagnostics or in biotechnology. In addition, they may be involved in pharmaceutical product manufacturing, product development, quality assurance, or pharmaceutical consulting.
Molecular biology or molecular genetics deals with the study of the structure and biosynthesis of nucleic acids and the processes associated with the transfer and implementation of this information in the form of proteins. This makes it possible to understand the painful dysfunctions of these functions and, possibly, to cure them with gene therapy. There are interfaces for biotechnology and genetic engineering in which simple organisms such as bacteria and yeast are created to make substances of pharmacological or commercial interest commercially available through targeted mutations.
The pharmaceutical and chemical industry offers numerous areas of employment for molecular biologists. In an industrial setting, they analyze biotransformation processes or develop and improve processes for the microbiological production of active ingredients and pharmaceutical intermediates. In addition, they are involved in the transition of newly developed products from research to production. In carrying out verification tasks, they ensure that manufacturing facilities, equipment, analytical methods and all steps in the production of sensitive products such as pharmaceuticals always meet the required quality standards. In addition, molecular biologists advise users on the use of new products.
A master's program is often required for leadership positions.
In the field of science and research, molecular biologists deal with topics such as the recognition, transport, folding, and codification of proteins in the cell. Research results, which form the basis for practical application in various fields, are published and thus made available to other scientists and students. At conferences and congresses, they discuss and present the results of scientific activities. Molecular biologists give lectures and seminars, direct scientific work and take exams.
Independent research activities require a master's and doctoral degrees.
1. Introduction.
Subject, tasks and methods of molecular biology and genetics. The value of "classical" genetics and genetics of microorganisms in the development of molecular biology and genetic engineering. The concept of a gene in "classical" and molecular genetics, its evolution. Contribution of genetic engineering methodology to the development of molecular genetics. Applied value of genetic engineering for biotechnology.
2. Molecular basis of heredity.
The concept of a cell, its macromolecular composition. The nature of the genetic material. History of proof of the genetic function of DNA.
2.1. Various types of nucleic acids. Biological functions of nucleic acids. Chemical structure, spatial structure and physical properties of nucleic acids. Features of the structure of the genetic material of pro - and eukaryotes. Complementary Watson-Crick base pairs. Genetic code. The history of decoding the genetic code. The main properties of the code: tripletness, code without commas, degeneracy. Features of the code dictionary, families of codons, semantic and "nonsense" codons. Circular DNA molecules and the concept of DNA supercoiling. DNA topoisomers and their types. Mechanisms of action of topoisomerases. DNA gyrase of bacteria.
2.2. DNA transcription. RNA polymerase of prokaryotes, its subunit and three-dimensional structures. Variety of sigma factors. Prokaryotic gene promoter, its structural elements. Stages of the transcriptional cycle. Initiation, formation of an "open complex", elongation and termination of transcription. Attenuation of transcription. Regulation of the expression of the tryptophan operon. "Ribo switches". Transcription termination mechanisms. Negative and positive regulation of transcription. Lactose operon. Regulation of transcription in the development of the lambda phage. Principles of DNA recognition by regulatory proteins (CAP protein and lambda phage repressor). Features of transcription in eukaryotes. RNA processing in eukaryotes. Capping, splicing and polyadenylation of transcripts. Splicing mechanisms. The role of small nuclear RNAs and protein factors. Alternative splicing, examples.
2.3. Broadcast, its stages, the function of ribosomes. Localization of ribosomes in the cell. Prokaryotic and eukaryotic types of ribosomes; 70S and 80S ribosomes. Morphology of ribosomes. Subdivision into subparticles (subunits). Codon-dependent binding of aminoacyl-tRNA in the elongation cycle. Codon-anticodon interaction. Involvement of the elongation factor EF1 (EF-Tu) in the binding of aminoacyl-tRNA to the ribosome. Elongation factor EF1B (EF-Ts), its function, sequence of reactions with its participation. Antibiotics affecting the stage of codon-dependent binding of aminoacyl-tRNA to the ribosome. Aminoglycoside antibiotics (streptomycin, neomycin, kanamycin, gentamicin, etc.), their mechanism of action. Tetracyclines as inhibitors of the binding of aminoacyl-tRNA to the ribosome. Broadcast initiation. The main stages of the initiation process. Initiation of translation in prokaryotes: initiation factors, initiation codons, 3 -end of RNA of the small ribosomal subunit and the Shine-Dalgarno sequence in mRNA. Initiation of translation in eukaryotes: initiation factors, initiation codons, 5 ¢ untranslated region, and cap-dependent “terminal” initiation. "Internal" cap-independent initiation in eukaryotes. Transpeptidation. Transpeptidation inhibitors: chloramphenicol, lincomycin, amycetin, streptogramins, anisomycin. Translocation. Involvement of the elongation factor EF2 (EF-G) and GTP. Translocation inhibitors: fusidic acid, viomycin, their mechanisms of action. Broadcast termination. Termination codons. Protein termination factors for prokaryotes and eukaryotes; two classes of termination factors and their mechanisms of action. Regulation of translation in prokaryotes.
2.4. DNA replication and its genetic control. Polymerases involved in replication, characteristics of their enzymatic activities. Accuracy of DNA reproduction. The role of steric interactions between DNA base pairs during replication. E. coli polymerases I, II and III. Polymerase III subunits. Replication fork, master and lag threads in replication. Fragments of Okazaki. A complex of proteins in a replication fork. Regulation of replication initiation in E. coli. Termination of replication in bacteria. Features of the regulation of plasmid replication. Bi-directional and rolling ring replication.
2.5. Recombination, its types and models. General or homologous recombination. Double-strand DNA breaks initiating recombination. The role of recombination in post-replicative repair of double-strand breaks. Holliday structure in the recombination model. Enzymology of general recombination in E. coli. RecBCD complex. RecA protein. The role of recombination in ensuring DNA synthesis in case of DNA damage that interrupts replication. Recombination in eukaryotes. Recombination enzymes in eukaryotes. Site-specific recombination. Differences in the molecular mechanisms of general and site-specific recombination. Recombinase classification. Types of chromosomal rearrangements carried out during site-specific recombination. The regulatory role of site-specific recombination in bacteria. Construction of chromosomes of multicellular eukaryotes using a site-specific phage recombination system.
2.6. DNA repair. Classification of types of repair. Direct repair of thymine dimers and methylated guanine. Cutting out the bases. Glycosylase. The mechanism of repair of unpaired nucleotides (mismatch repair). Selection of the DNA strand to be repaired. SOS repair. Properties of DNA polymerases involved in SOS repair in prokaryotes and eukaryotes. The concept of "adaptive mutations" in bacteria. Repair of double-strand breaks: homologous post-replicative recombination and fusion of non-homologous ends of the DNA molecule. The relationship between the processes of replication, recombination and repair.
3. Mutational process.
The Role of Biochemical Mutants in Forming the One Gene - One Enzyme Theory. Classification of mutations. Point mutations and chromosomal rearrangements, the mechanism of their formation. Spontaneous and induced mutagenesis. Classification of mutagens. Molecular mechanism of mutagenesis. The relationship between mutagenesis and repair. Identification and selection of mutants. Suppression: intragenic, intergenic and phenotypic.
4. Extrachromosomal genetic elements.
Plasmids, their structure and classification. Sexual factor F, its structure and life cycle. The role of factor F in the mobilization of chromosomal transfer. Formation of donors like Hfr and F ". Mechanism of conjugation. Bacteriophages, their structure and life cycle. Virulent and moderate bacteriophages. Lysogeny and transduction. General and specific transduction. Migrating genetic elements: transposons and IS-sequences, their role in genetic exchange. DNA -transposons in the genomes of prokaryotes and eukaryotes IS-sequences of bacteria, their structure IS-sequences as a component of the F-factor of bacteria, which determines the ability to transfer genetic material during conjugation Transposons of bacteria and eukaryotic organisms Direct non-replicative and replicative mechanisms of transpositions Concept of horizontal transfer of transposons and their role in structural rearrangements (ectopic recombination) and in genome evolution.
5. Study of the structure and function of the gene.
Elements of genetic analysis. Cis-trans complementation test. Genetic mapping using conjugation, transduction and transformation. Building genetic maps. Subtle genetic mapping. Physical analysis of the structure of the gene. Heteroduplex analysis. Restriction analysis. Sequencing methods. Polymerase chain reaction. Identifying gene function.
6. Regulation of gene expression. Operon and Regulon concepts. Control at the level of transcription initiation. Promoter, operator and regulatory proteins. Positive and negative control of gene expression. Control at the level of transcription termination. Catabolite-controlled operons: models of lactose, galactose, arabinose and maltose operons. Attenuator-controlled operons: a model of the tryptophan operon. Multivalent regulation of gene expression. Global systems of regulation. Regulatory response to stress. Post-transcriptional control. Sigal transduction. RNA-mediated regulation: small RNAs, sensory RNAs.
7. Basics of genetic engineering. Restriction and modification enzymes. Isolation and cloning of genes. Vectors for molecular cloning. Principles for the construction of recombinant DNA and their introduction into recipient cells. Applied aspects of genetic engineering.
1. Watson J., Ace J., Recombinant DNA: A Short Course. - M .: Mir, 1986.
2. Genes. - M .: Mir. 1987.
3. Molecular biology: structure and biosynthesis of nucleic acids. / Ed. ... - M. Higher school. 1990.
4., - Molecular biotechnology. M. 2002.
5. Spirin ribosomes and protein biosynthesis. - M .: Higher school, 1986.
1. Khesin of the genome. - M .: Science. 1984.
2. Rybchin genetic engineering. - SPb .: SPbSTU. 1999.
3. Patrushev of genes. - M .: Nauka, 2000.
4. Modern microbiology. Prokaryotes (in 2 volumes). - M .: Mir, 2005.
5. M. Singer, P. Berg. Genes and genomes. - M .: Mir, 1998.
6. Nutcrackers engineering. - Novosibirsk: From Sib. Univ., 2004.
7. Stepanov biology. The structure and function of proteins. - M .: V. Sh., 1996.
Molecular biology / m ə lɛ ToJʊ lər / is a branch of biology that deals with the molecular basis of biological activity between biomolecules in various cell systems, including the interactions between DNA, RNA, proteins and their biosynthesis, as well as the regulation of these interactions. Enrollment in nature in 1961, Astbury described molecular biology:
Not so much a technique as an approach, an approach from the point of view of the so-called fundamental sciences with the leading idea of searching below the large-scale manifestations of classical biology for the corresponding molecular plan. It is concerned, in particular, with forms biological molecules and [...] predominantly three-dimensional and structural - which does not mean, however, that this is just a refinement of morphology. He must at the same time explore genesis and function.
Researchers in the field of molecular biology use specific methods of outgrowing molecular biology, but more and more combine them with methods and ideas from genetics and biochemistry. There is no definite line between these disciplines. This is illustrated in the following diagram, which depicts one possible kind of relationship between fields:
One of the most basic molecular biology techniques for studying protein function is molecular cloning. In this technique, DNA encoding a protein of interest is cloned by polymerase chain reaction (PCR) and / or restriction enzymes in a plasmid (expression vector). The vector has 3 distinctive features: an origin of replication, a multiple cloning site (MCS), and a selectable marker, usually antibiotic resistant. The upstream multiple cloning sites are promoter regions and transcriptional initiation sites that regulate expression of the cloned gene. This plasmid can be inserted into either bacterial or animal cells. The introduction of DNA into bacterial cells can be done by transformation using naked DNA uptake, conjugation using cell-cell contacts, or by transduction using a viral vector. The introduction of DNA into eukaryotic cells, such as animal cells, by physical or chemical means, is called transfection. Several different transfection methods are available, such as calcium phosphate transfection, electroporation, microinjection, and liposomal transfection. The plasmid can be integrated into the genome, resulting in stable transfection, or it can remain independent of the genome, called transient transfection.
DNA encoding proteins of interest are now inside the cell, and proteins can now be expressed. Diverse systems such as inducible promoters and specific cellular signaling factors help express protein interest at high levels. Large amounts of protein can then be recovered from the bacterial or eukaryotic cell. A protein can be tested for enzymatic activity in various situations, the protein can be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.
Terms northern , west and Oriental blotting gets from what was originally molecular biology a joke that played on the term Southernnet, following the procedure described by Edwin Southern for BLOTTED DNA hybridization. Patricia Thomas, developer of RNA blotting, which then became known as north - blotting, don't really use that term.
Named after its inventor, biologist Edwin South, the Southern blot is a method for examining the presence of a specific DNA sequence in a DNA sample. DNA samples before or after restriction enzyme (restriction enzyme) digestions are separated by gel electrophoresis and then transferred to the membrane by blotting using capillary action. The membrane is then exposed to a labeled DNA probe that has a base sequence complementary to that on the DNA of interest. Southern blotting is less widely used in the scientific laboratory due to the ability of other methods, such as PCR, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring a transgene copy number in transgenic mice or in gene engineering of knockout embryonic stem cell lines.
Northern blot chart
Clinical research and medical therapies arising from molecular biology are partially covered by gene therapy. The application of molecular biology or molecular cell biology approaches in medicine is now called molecular medicine. Molecular biology also plays an important role in understanding the formation, actions and regulations of various parts of cells, which can be used to effectively target new drugs, diagnose disease, and understand cell physiology.
Molecular biology has experienced a period of rapid development of its own research methods, which now distinguish it from biochemistry. These include, in particular, methods of genetic engineering, cloning, artificial expression and gene knockout. Since DNA is a 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 branch of genetics and molecular biology. Just as molecular biology widely uses viruses as a research tool, in virology, methods of molecular biology are used to solve their problems. Computer technology is involved in the analysis of genetic information, and therefore new areas of molecular genetics have appeared, which are sometimes considered special disciplines: bioinformatics, genomics, and proteomics.
This fundamental discovery was prepared by a long phase of research into the genetics and biochemistry of viruses and bacteria.
In 1928, Frederick Griffith showed for the first time that an extract of heat-killed pathogenic bacteria could transmit pathogenicity to non-hazardous bacteria. The study of the transformation of bacteria later led to the purification of the pathogenic agent, which, contrary to expectations, turned out to be not a protein, but a nucleic acid. By itself, the nucleic acid is not dangerous, it only transfers 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, like transformation, formed the basis of plasmid technology widespread in molecular biology. Another important discovery for methodology was the discovery of bacterial viruses and bacteriophages at the beginning of the 20th century. Phages can also transfer genetic material from one bacterial cell to another. Infection of bacteria with 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 the RNA becomes more similar to the DNA of a bacteriophage. 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. So 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. Senger, Nobel Prize in Chemistry, 1980), and new discoveries in the field of studies of 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 most important for medicine, agriculture and scientific research, which led to the emergence of several new directions in biology: genomics, bioinformatics, etc.
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Advances in the study of nucleic acids and protein biosynthesis have led to the creation of a number of methods that are of great applied importance in medicine, agriculture, and a number of other industries.
After the genetic code and the basic principles of storing and realizing hereditary information were studied, the development of molecular biology came to a dead end, since there were no methods that could manipulate genes, isolate and change them. The emergence of these methods took place in the 1970s and 1980s. This gave a powerful impetus to the development of this field of science, which is still flourishing today. First of all, these methods relate to the production of individual genes and their introduction into cells of other organisms (molecular cloning and transgenesis, PCR), as well as methods for determining the sequence of nucleotides 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 advanced methods.
It is a basic DNA technique that is used in conjunction with almost all other methods to isolate the desired molecules and analyze the results. To separate DNA fragments by length, the method of gel electrophoresis is used. DNA is an acid, its molecules contain phosphoric acid residues, which split off a proton and acquire a negative charge (Fig. 1).
Therefore, in an electric field, DNA molecules move to the anode - a positively charged electrode. This occurs in an electrolyte solution containing charge carrier ions, so that this solution conducts current. To separate the fragments, a dense polymer gel (agarose or polyacrylamide) is used. DNA molecules "get entangled" 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 of a sample, they are compared with a marker - a set of fragments of standard lengths applied in parallel on the same gel (Fig. 4).
The most important tools for working with DNA are enzymes that transform DNA in living cells: DNA polymerases, DNA ligases and restriction endonucleases, or restriction enzymes. DNA polymerase carry out matrix synthesis of DNA, which allows DNA to be multiplied in a test tube. DNA ligases stitch together DNA molecules or heal gaps in them. Restriction endonucleases, or restriction enzymes, 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 separate genes.
The sequences recognized by restriction endonucleases are symmetrical, and breaks can occur in the middle of such a sequence or with a shift (at the same place in both DNA strands). The scheme of action of different types of restriction enzymes 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 turns out to be shorter than the other; a single-stranded section is formed with a symmetrical sequence identical at both ends formed.
The terminal sequences will be the same when any DNA is cleaved with a given restriction enzyme and can be re-linked since they have complementary sequences. They can be stitched together using DNA ligase and get a single molecule. Thus, it is possible to combine fragments of two different DNA and obtain the so-called recombinant DNA... This approach is used in the method of molecular cloning, which allows you to obtain individual genes and introduce them into cells that can form a protein encoded in the gene.
Molecular cloning uses two DNA molecules - an insert containing the gene of interest, and vector- DNA serving as a carrier. The insert is "sewn" into the vector using enzymes to obtain 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 the offspring of one cell, that is, a clone, all cells in a colony are genetically identical and contain the same recombinant DNA. Hence the term "molecular cloning", that is, obtaining a clone of cells containing the DNA fragment of interest to us. After the colonies containing the insert of interest to us are obtained, it is possible to characterize this insert by various methods, for example, to determine its exact sequence. Cells can also produce an insert-encoded protein if it contains a functional gene.
When a recombinant molecule is introduced into cells, 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 incorporation into the genome, which leads to the appearance in such a cell of new inherited traits characteristic of a DNA donor organism. For example, if the inserted molecule contains the 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 trait appears in the transformed cells.
A vector must have a number of properties:
First, it is a relatively small DNA molecule to be easily manipulated.
Second, in order for DNA to be preserved and multiplied in a cell, it must contain a certain sequence that ensures its replication (the origin of replication, or origin of replication).
Third, it must contain gene marker, which ensures the selection of only those cells into which the vector has fallen. 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, since they are easy to cultivate and multiply quickly. A bacterial cell usually contains one large circular DNA molecule, several million nucleotide 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 sequence of nucleotides that ensure 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 to distinguish plasmid-bearing cells from normal cells. Plasmids that carry two genes that provide resistance to two antibiotics, for example, tetracycline and amicillin, are more commonly used. There are simple methods for isolating such plasmid DNAs free from the DNA of the bacterial main chromosome.
The transfer of genes from one organism to another is called transgenesis, and such modified organisms - transgenic... By transferring genes into the cells of microorganisms, recombinant protein preparations for the needs of medicine are obtained, in particular, human proteins that do not cause immune rejection - interferons, insulin and other protein hormones, cellular growth factors, as well as proteins for the production of vaccines. In more complex cases, when the modification of proteins proceeds correctly only in eukaryotic cells, transgenic cell cultures or transgenic animals are used, in particular, livestock (primarily goats), which secretes the necessary proteins into milk, or proteins are isolated from their blood. This is how antibodies, clotting factors and other proteins are obtained. By the method of transgenesis, crop plants are obtained that are resistant to herbicides and pests and have other useful properties. With the help of transgenic microorganisms, they 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 methods of modification and gene transfer.
molecular cloning technology
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 make 1 break in the plasmid DNA in one of the resistance genes, for example, to ampicillin.
The molecular cloning process includes the following steps:
Cutting and stitching - 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 have received an insert vector.
Plasmid DNA is treated with the same restriction enzymes, and it is converted into a linear molecule if such a restriction enzyme is selected that introduces 1 gap into the plasmid. As a result, the ends of all the resulting DNA fragments end up with the same sticky ends. When the temperature is lowered, these ends are connected randomly, and they 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 - fragments of chromosomal DNA joined together, and still others - a reduced circular plasmid or its dimer (Fig. 4).
Then 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 incorporation into the genome, which leads to the appearance in such a cell of new inherited traits characteristic of a DNA donor organism. 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).
Next, the task is to select only the cells in which the vector with the insertion fell, and to distinguish them from the cells that carry only the vector without the insertion or do not carry the vector at all. This process of selecting the desired cells is called breeding... For this, use selective markers- usually antibiotic resistance genes in the vector, and selective media containing antibiotics or other substances that provide selection.
In our example, cells from colonies grown in the presence of ampicillin are subcultured into 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 on the medium with tetracycline will not grow (Fig. 5). Among them, by special methods, those that contain the gene of interest to us are selected, grown in sufficient quantities and plasmid DNA is isolated. From it, using the same restriction enzymes that were used to obtain recombinant DNA, the individual gene of interest is excised. The DNA of this gene can be used to determine the sequence of nucleotides, to introduce it into an organism to obtain new properties, or to synthesize the desired protein. This method of isolating genes is called molecular cloning.
It is very convenient to use fluorescent proteins as marker genes in studies of eukaryotic organisms. Gene of 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 use of this protein.
Then the genes of other fluorescent proteins were isolated - red, blue, yellow. These genes have been artificially modified to produce proteins with the desired properties. The variety 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 ligated with the gene of any other protein, then during translation a single protein will be formed - a translationally fusion protein, or fusion(fusion protein) that fluoresces. Thus, it is possible to study, for example, the localization (location) of any proteins of interest in the cell, their movement. By expressing fluorescent proteins only in certain types of cells, it is possible to mark cells of these types in a multicellular organism (see Fig. 8 - a mouse brain, in which individual neurons have different colors due to a certain combination of genes of fluorescent proteins). Fluorescent proteins are an indispensable tool in modern molecular biology.
Another method of 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 it happens in cells during DNA replication.
The origin of replication in this method is defined by two small pieces of DNA called seeds, or primers... These primers are complementary to the ends of the gene of interest on two DNA strands. First, the chromosomal DNA, from which the gene must be isolated, is mixed with seeds and heated to 99 ° C. This leads to the rupture of hydrogen bonds and the divergence of DNA strands. Thereafter, the temperature is lowered to 50-70 ° C (depending on the length and sequence of the seeds). Under these conditions, the primers attach to the complementary regions of the chromosomal DNA, forming a regular double helix (see Fig. 9). After that, a mixture of all four nucleotides required for DNA synthesis and DNA polymerase are added. The enzyme lengthens the primers by building double-stranded DNA from where the primers are attached, i.e. from the ends of the gene to the end of the single-stranded chromosomal molecule. 
If the mixture is now heated again, the chromosomal and newly synthesized chains will disperse. After cooling, they will again be joined by seeds, which are taken in large excess (see Fig. 10).
On the newly synthesized chains, they will attach not to the end from which the first synthesis began, but to the opposite end, 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, capable of withstanding boiling and operating 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 in each cycle the number of sequences bounded at both ends by the injected seeds (see Fig. 12).
After about 25 such cycles, the copy number of the gene will increase 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.
Another important achievement is the development of methods for determining the sequence of nucleotides in DNA - DNA sequencing(from the English sequence - sequence). To do this, it is necessary to obtain genes pure from other DNA by one of the described methods. Then the DNA strands are separated by heating and a primer labeled with radioactive phosphorus or fluorescent label is added to them. Note that one seed is taken, complementary to one strand. 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 at the third atom of deoxyribose. If such a nucleotide is included in the synthesized DNA strand, then its lengthening will not be able to continue, because the polymerase will have nowhere to attach the next nucleotide. Therefore, DNA synthesis is terminated after the inclusion of such a nucleotide. Much less of such nucleotides, called dideoxynucleotides, are added than ordinary ones, so chain termination occurs only occasionally and in each chain in different places. The result is a mixture of chains of different lengths, with the same nucleotide at the end of each. 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 there was adenine in the gene in the second, seventh and twelfth positions. The resulting mixture of chains can be easily separated by size using electrophoresis, and the synthesized chains can be identified by radioactivity on an X-ray film (see Fig. 10).
It turns out the picture shown at the bottom of the figure, called a radio autograph. Moving along it from bottom to top and reading the letter above the columns of each zone, we get the nucleotide sequence shown in the figure to the right of the autograph. It turned out that the 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 marked with its own dye, then the zones obtained during the separation of the synthesized strands will glow with different light. This makes it possible to carry out the reaction in one test tube simultaneously for all nucleotides and by dividing the obtained chains by length, to identify 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 the sequences of nucleotides in genes have now been developed. If the pair 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 makes it possible to predict a person's predisposition to many diseases and to take measures in advance to avoid them.
