Molecular genetics

Molecular genetics is the field of biology that studies the structure and function of genes at a molecular level and thus employs methods of both molecular biology and genetics. The study of chromosomes and gene expression of an organism can give insight into heredity, genetic variation, and mutations. This is useful in the study of developmental biology and in understanding and treating genetic diseases.

Techniques

Amplification

Gene amplification is a procedure in which a certain gene or DNA sequence is replicated many times in a process called DNA replication.

Polymerase chain reaction
The main genetic components of the polymerase chain reaction (PCR) are DNA nucleotides, template DNA, primers and Taq polymerase. DNA nucleotides make up the DNA template strand for the specific sequence being amplified, primers are short strands of complementary nucleotides where DNA replication starts, and Taq polymerase is a heat stable enzyme that jump-starts the production of new DNA at the high temperatures needed for reaction.[1]
Cloning DNA in bacteria
Cloning is the process of creating many identical copies of a sequence of DNA. The target DNA sequence is 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"[2] and create a recombinant DNA molecule.

The recombinant DNA molecules are then put into a bacterial 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 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 as 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.[3]
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 isolating 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]

Genetic screens

Forward genetics

This technique is used to identify which genes or genetic mutations produce a certain phenotype. A mutagen is very often used to accelerate this process. Once mutants have been isolated, the mutated genes can be molecularly identified.

Forward saturation genetics is a method for treating organisms with a mutagen, then screens the organism's offspring for particular phenotypes. This type of genetic screening is used to find and identify all the genes involved in a trait.[5]

Reverse genetics
Reverse genetics determines the phenotype that results from a specifically engineered gene. In some organisms, such as yeast and mice, it is possible to induce the deletion of a particular gene, creating what's known as a gene "knockout" - the laboratory origin of so-called "knockout mice" for further study. In other words this process involves the creation of transgenic organisms that do not express a gene of interest. Alternative methods of reverse genetic research include the random induction of DNA deletions and subsequent selection for deletions in a gene of interest, as well as the application of RNA interference.

Gene therapy

A mutation in a gene can cause encoded proteins and the cells that rely on those proteins to malfunction. Conditions related to gene mutations are called genetic disorders. However, altering a patient's genes can sometimes be used to treat or cure a disease as well. Gene therapy can be used to replace a mutated gene with the correct copy of the gene, to inactivate or knockout the expression of a malfunctioning gene, or to introduce a foreign gene to the body to help fight disease.[6] Major diseases that can be treated with gene therapy include viral infections, cancers, and inherited disorders, including immune system disorders.[7]

Gene therapy delivers a copy of the missing, mutated, or desired gene via a modified virus or vector to the patient's target cells so that a functional form of the protein can then be produced and incorporated into the body.[8] These vectors are often siRNA.[9] Treatment can be either in vivo or ex vivo. The therapy has to be repeated several times for the infected patient to continually be relieved, as repeated cell division and cell death slowly randomizes the body's ratio of functional-to-mutant genes. Gene therapy is an appealing alternative to some drug-based approaches, because gene therapy repairs the underlying genetic defect using the patients own cells with minimal side effects.[9] Gene therapies are still in development and mostly used in research settings. All experiments and products are controlled by the U.S. FDA and the NIH. [10][11]

Classical gene therapies usually require efficient transfer of cloned genes into the disease cells so that the introduced genes are expressed at sufficiently high levels to change the patient's physiology. 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 is 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.[12]

The Human Genome Project

The Human Genome Project is a molecular genetics project that began in the 1990s 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:

  1. identifying 20,000 to 25,000 genes in human DNA (although initial estimates were approximately 100,000 genes),
  2. determining sequences of chemical base pairs in human DNA,
  3. storing all found information into databases,
  4. improving the tools used for data analysis,
  5. transferring technologies to private sectors, and
  6. addressing the ethical, legal, and social issues (ELSI) that may arise from the projects.[13]

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.[13]

See also

Sources and notes

  1. ↑ Ramsden, Jeremy J (2009). Bioinformatics: An Introduction. New York: Springer. p. 191. ISBN 978-1-84800-256-2.
  2. ↑ NCBI
  3. ↑ "DNA isolation methods" (PDF). Archived from the original (PDF) on March 18, 2015.
  4. ↑
  5. ↑ "Forward and Reverse Genetics" (PDF).
  6. ↑ Reference, Genetics Home. "What is gene therapy?".
  7. ↑ "Search of: "gene therapy" - List Results - ClinicalTrials.gov".
  8. ↑ Berg, Jeremy M., John L. Tymoczko, and Lubert Stryer. "Chapter 5: Exploring Genes and Genomes." Biochemistry. 7th ed. New York City: W.H. Freeman, 2012. N. pag. Print.
  9. 1 2 Herrera-Carrillo E, Berkhout B. Bone Marrow Gene Therapy for HIV/AIDS. Viruses 2015;7(7):3910-36.
  10. ↑ Reference, Genetics Home. "Is gene therapy available to treat my disorder?".
  11. ↑ Reference, Genetics Home. "Is gene therapy safe?".
  12. ↑
  13. 1 2 "Human Genome Project Information".

Further reading

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