Molecular genetics is the field of biology which studies the structure and function of genes at a molecular level. Foundations of modern biology There are five unifying principles History See also History of genetics The existence of genes was first suggested by Gregor Mendel (1822-1884 who in the 1860s studied inheritance In Chemistry, a molecule is defined as a sufficiently stable electrically neutral group of at least two Atoms in a definite arrangement held together by The field studies how the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology. Genetics (from Ancient Greek grc-Latn genetikos, “genitive” and that from grc-Latn genesis, “origin” a discipline of Biology, is Molecular biology is the study of Biology at a molecular level It is so-called to differentiate it from other sub fields of genetics such as ecological genetics and population genetics. Ecological genetics is the study of Genetics in the context of the interactions among organisms and between the organisms and their environment Population genetics is the study of the Allele frequency distribution and change under the influence of the four evolutionary forces Natural selection, Genetic 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. A genetic screen (often shortened to screen) is a procedure or test to identify and select individuals who possess a Phenotype of interest The aim of this technique is to identify mutations that produce a certain phenotype. A phenotype is any observable characteristic of an Organism, such as its morphology, Development, biochemical or physiological properties A mutagen is very often used to accelerate this process. In Biology, a mutagen ( Latin, literally origin of change) is a physical or chemical agent that changes the genetic information (usually DNA) Once mutants have been isolated, the mutated gene can be molecularly identified. In biology mutations are changes to the Nucleotide sequence of the Genetic material of an organism
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. Reverse genetics is an approach to discovering the function of a Gene that proceeds in the opposite direction of so called forward genetic screens of Classical 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. A knockout mouse is a genetically engineered mouse in which one or more Genes have been turned off through a Gene knockout. A gene knockout is a genetic technique in which an organism is engineered to carry genes that have been made inoperative (have been "knocked out" of the organism 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. RNA interference ( RNAi) is a mechanism that inhibits Gene expression at the stage of translation or by hindering the transcription of specific This article is about organisms which have been genetically modified
Gene Therapy
Many mutations in a person's genes result in severe medical condition. Gene therapy is the insertion of Genes into an individual's cells and tissues to treat a Disease, and Hereditary diseases in which a 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 and this can be the only possible option in patients with tissues where individual cells cannot be cultured invitro, 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 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 polymerase chain reaction. Cloning DNA in bacteria is also a way to amplify DNA in genes.
Polymerase chain reaction
The main materials used in 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. Cell culture is the process by which prokaryotic, or eukaryotic cells are grown under controlled 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). Messenger ribonucleic acid ( mRNA) is a molecule of RNA encoding a chemical "blueprint" for a Protein product 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). Adenine is a Purine with a variety of roles in Biochemistry including Cellular respiration, in the form of both the energy-rich Adenosine 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. Thymine is one of the four bases in the Nucleic acid of DNA that make up the letters ATGC 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. In Biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase Enzyme that transcribes A DNA Polymerase is an Enzyme that assists in DNA replication. 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. In Genetics, complementary DNA ( cDNA) is DNA synthesized from a mature MRNA template in a reaction catalyzed by the enzyme Reverse [4]
The Molecular Genetics Project
The Human Genome Project is a molecular genetics project that began in the 1980's and was projected to take fifteen years to complete. The Human Genome Project (HGP was an international Scientific research project with a primary goal to determine the sequence of chemical base pairs which make up DNA 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]
See also
- Human Genome Project
- The study of macromolecules important in biological inheritance
- Forward and reverse genetics screens
- Transgenics and overexpression
- Mapping, cloning, and sequencing
- Gene expression
- Measuring
- Spatial and temporal regulation
- cDNAs
- Imprinting
- Bacterial and phage molecular genetics
- Eukaryotic molecular genetics
Sources and notes
Further reading
- Atlas of Genetics and Cytogenetics in Oncology and Haematology [1]
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