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Molecular genetics

Molecular genetics is the field of biology which studies the structure and function of genes at a molecular level. The field studies how the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology. It is so-called to differentiate it from other sub fields of genetics such as ecological genetics and population genetics. An important area within molecular genetics is the use of molecular information to determine the patterns of descent, and therefore the correct scientific classification of organisms: this is called molecular systematics.

Along with determining the pattern of descendants, molecular genetics helps in understanding genetic mutations that can cause certain types of diseases. Through utilizing the methods of genetics and molecular biology, molecular genetics discovers the reasons why traits are carried on and how and why some may mutate.

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Forward genetics

One of the first tools available to molecular geneticists is the forward genetic screen. The aim of this technique is to identify mutations that produce a certain phenotype. A mutagen is very often used to accelerate this process. Once mutants have been isolated, the mutated gene can be molecularly identified.

Reverse genetics

Main article: Reverse genetics

While forward genetic screens are productive, a more straightforward approach would be to determine the phenotype that results from mutating a given gene. This is called reverse genetics. In some organisms, such as yeast and mice, it is possible to induce the deletion of a particular gene, creating a gene knockout. Alternatives include the random induction of DNA deletions and subsequent selection for deletions in a gene of interest, the application of RNA interference and the creation of transgenic organisms that do not express a gene of interest.

Gene Therapy

Main article: Gene therapy

Many mutations in a person's genes result in severe medical condition. These mutations cause the protein encoded by that gene to malfunction and cells that rely on this protein cannot function properly. This can cause problems for the tissues or organs. Conditions related to gene mutations are called genetic disorders. One way to fix the internal problems is gene therapy. By adding a corrected copy of the gene, the affected cells, tissues, and organs may work properly. As opposed to drug-based approaches, gene therapy repairs the underlying genetic defect.

Gene Therapy is the process to treat or alleviate diseases by genetically modifying the cells of the infected person, causing the gene to function properly. When a human disease gene has been recognized, molecular genetics tools can be used to explore the process of the gene in both the normal and pathogenic states. From there, the gene is transferred either in vivo or ex vivo and the body begins to identify the new gene. Gene therapy has to be repeated several times for the infected patient to continually be relieved.

Currently, gene therapy is still being experimented with and products are not approved by the U.S. Food and Drug Administration. There have been several set backs in the last 15 years that have restricted further measures on gene therapy. As there are unsuccessful attempts, there continues to be a growing number of successful gene therapy transfers which has furthered the research.

Major diseases that can be treated with gene therapy: infectious diseases (result of infection by a virus or bacterial pathogen), cancers, inherited disorders, immune system disorders

Classical gene therapy

Classical gene therapy is the approach which delivers genes to the appropriate target cells with a goal of attaining optimal expression of the new, introduced gene. Once inside the patient, the expressed genes are intended to: produce a product that the patient lacks, kill the diseased cells directly by producing a toxin, and activate the immune system to help the killing of the diseased cells.

Nonclassical gene therapy

Nonclassical gene therapy inhibits the expression of genes related to pathogenesis, or corrects a genetic defect and restores normal gene expression.

In vivo gene transfer

During In vivo gene transfer the genes are transferred directly into the tissue of the patient. This can be the only possible option in patients with tissues where individual cells cannot be cultured outside the body in sufficient numbers (i.e. brain cells). Also, in vivo gene transfer is necessary when cultured cells cannot be re-implanted in patients effectively.

Ex vivo gene transfer

During Ex vivo gene transfer the cells are cultured outside the body and then the genes are transferred into the cells grown in culture. The cells that have been transformed successfully are expanded by cell culture and then introduced into the patient.

Principles for gene transfer

Classical gene therapies usually require efficient transfer of cloned genes into the disease cells to the introduced genes are expressed at high levels. There are several different physicochemical and biological methods that can be used to transfer genes into human cells. The size of the DNA fragments that can be transferred are very limited, and often the transferred gene is not a conventional gene. Horizontal gene transfer is the transfer of genetic material from one cell to another that is not its offspring. Artificial horizontal gene transfer is a form of genetic engineering.^[1]

Techniques in Molecular Genetics

There are three general techniques used for molecular genetics: amplification, separation and detection, and expression. Specifically used for amplification is the Polymerase chain reaction, which is an “indispensable tool in a great variety of applications”^[2]. In the separation and detection technique DNA and mRNA are isolated from their cells. Gene expression in cells or organisms is done in a place or time that is not normal for that specific gene.

Amplification

There are other methods for amplification besides the Polymerase Chain Reaction. Cloning DNA in bacteria is also a way to amplify DNA in genes.

Polymerase Chain Reaction

The main materials used in the Polymerase Chain Reaction are DNA nucleotides, template DNA, primers and Taq polymerase. DNA nucleotides are the base for the new DNA, the template DNA is the specific sequence being amplified, primers are complementary nucleotides that can go on either side of the template DNA, and Taq polymerase is a heat stable enzyme that jump-starts the production of new DNA. This technique does not need to use living bacteria or cells, all that is needed is the base sequence of the DNA needing amplification.

Cloning DNA in Bacteria

The word cloning for this type of amplification entails making multiple identical copies of a sequence of DNA. The target DNA sequence is then inserted into a [cloning vector]. Because this vector originates from a self-replicating virus, plasmid, or higher organism cell when the appropriate size DNA is inserted the “target and vector DNA fragments are then ligated”^[3] and create a recombinant DNA molecule. The recombinant DNA molecules are then put into a bacteria strain (usually E. coli) which produces several identical copies by transformation. Transformation is the DNA uptake mechanism possessed by bacteria. However, only one recombinant DNA molecule can be cloned within a single bacteria cell, so each clone is of just one DNA insert.

Separation and Detection

In separation and detection DNA and mRNA are isolated from cells (the separation) and then detected simply by the isolation. Cell cultures are also grown to provide a constant supply of cells ready for isolation.

Cell Cultures

A cell culture for molecular genetics is a culture that is grown in artificial conditions. Some cell types grow well in cultures such a skin cells, but other cells are not as productive in cultures. There are different techniques for each type of cell, some only recently being found to foster growth in stem and nerve cells. Cultures for molecular genetics are frozen in order to preserve all copies of the gene specimen and thawed only when needed. This allows for a steady supply of cells.

DNA Isolation

DNA isolation extracts DNA from a cell in a pure form. First, the DNA is separated from cellular components such as proteins, RNA, and lipids. This is done by placing the chosen cells in a tube with a solution that mechanically, chemically, breaks the cells open. This solution contains enzymes, chemicals, and salts that breaks down the cells except for the DNA. It contains enzymes to dissolve proteins, chemicals to destroy all RNA present, and salts to help pull DNA out of the solution.

Next, the DNA is separated from the solution by being spun in a centrifuge, which allows the DNA to collect in the bottom of the tube. After this cycle in the centrifuge the solution is poured off and the DNA is resuspended in a second solution that makes the DNA easy to work with in the future.

This results in a concentrated DNA sample that contains thousands of copies of each gene. For large scale projects such as sequencing the human genome, all this work is done by robots.

mRNA Isolation

Expressed DNA that codes for the synthesis of a protein is the final goal for scientists and this expressed DNA is obtained by isolation mRNA (Messenger RNA). First, laboratories use a normal cellular modification of mRNA that adds up to 200 adenine nucleotides to the end of the molecule (poly(A) tail). Once this has been added, the cell is ruptured and its cell contents are exposed to synthetic beads that are coated with thymine string nucleotides. Because Adenine and Thymine pair together in DNA, the poly(A) tail and synthetic beads are attracted to one another, and once they bind in this process the cell components can be washed away without removing the mRNA. Once the mRNA has been isolated, reverse transcriptase is employed to convert it to single-stranded DNA, from which a stable double-stranded DNA is produced using DNA polymerase. Complementary DNA (cDNA) is much more stable than mRNA and so, once the double-stranded DNA has been produced it represents the expressed DNA sequence scientists look for.^[4]

The Molecular Genetics Project

The Human Genome Project is a molecular genetics project that began in 1990 and was projected to take fifteen years to complete. However, because of technological advances the progress of the project was advanced and the project finished in 2003, taking only thirteen years. The project was started by the U.S. Department of Energy and the National Institutes of Health in an effort to reach six set goals. These goals included:

identifying 20,000 to 25,000 genes in human DNA (although initial estimate were approximately 100,000 genes),
determining sequences of chemical based pairs in human DNA,
storing all found information into databases,
improving the tools used for data analysis,
transferring technologies to private sectors, and
addressing the ethical, legal, and social issues (ELSI) that may arise from the projects.^[5]

The project was worked on by eighteen different countries including the United States, Japan, France, Germany, and the United Kingdom. The collaborative effort resulted in the discovery of the many benefits of molecular genetics. Discoveries such as molecular medicine, new energy sources and environmental applications, DNA forensics, and livestock breeding, are only a few of the benefits that molecular genetics can provide.^[5]

Sources and notes

^ *Human Molecular Genetics
- Learn Genetics
- Medem
^ NCBI
^ NCBI
^ *NCBI
- Molecular Techniques
^ ^a ^b Human Genome