Applications of molecular genetics

Application of recombinant dna

1. Molecular Genetics Topic of presentation : Applications of Molecular Genetics Presentor : Sadia Mazhar College roll no.880 University roll no.023809

2. Applications and techniques of Molecular Genetics Molecular Genetics: Molecular genetics  is the study of the  molecular  structure of DNA, its cellular activities (including its replication), and its influence in determining the overall makeup of an organism.  Molecular genetics  relies heavily on  genetic  engineering (recombinant DNA technology), which in medicine has been  used  to mass-produce insulin, human growth hormones, follistim (for treating infertility), human albumin, monoclonal antibodies, antihemophilic factors, vaccines, and many other drugs. In research, organisms are  genetically  engineered to discover the functions of certain  genes .

3. A Simple Example-Translational Proces

4. Techniques of Molecular Genetics Gene Therapy Amplification Polymerase Chain Reaction(PCR) Cell Culture DNA Cloning in Bacteria Gel Electrophoresis Isolation of DNA

5. Definition: Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery. Gene Therapy

6. Approaches of Gene Therapy Replacing a mutated gene that causes disease with a healthy copy of the gene. Inactivating, or “knocking out,” a mutated gene that is functioning improperly. Introducing a new gene into the body to help fight a disease.

7. Gene therapy is an experimental form of treatment that uses gene transfer of genetic material into the cell of a patient to cure the disease. The idea is to modify the genetic information of the cell of the patient that is responsible for a disease, and then return that cell to normal conditions. Transfer of genetic material is done commonly by using viral vectors that use their own biological capacities to enter the cell and deposit the genetic material. Both inherited genetic diseases and acquired disorders can be treated with gene therapy. Examples of these disorders are primary immune deficiencies, where gene therapy has been able to fully correct the presentation of patients, and/or cancer, where the gene therapy is still at the experimental stage.

8. Diseases treated by Gene Therapy With its potential to eliminate and prevent hereditary  diseases  such as cystic fibrosis and hemophilia and its use as a possible  cure  for heart  disease , AIDS, and cancer,  gene therapy  is a potential medical miracle-worker. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve your body's ability to fight disease. Gene therapy holds promise for treating a wide range of diseases, such as cancer , cystic fibrosis , heart disease , diabetes , hemophilia and AIDS .

9. Amplification In  molecular biology ,  amplification  is a process by which a nucleic acid  molecule  is enzymatically copied to generate a progeny population with the same sequence as the parental one. The most widely used  amplification  method is Polymerase Chain Reaction (PCR).  The result of a PCR amplification of a segment of   DNA  is called an “amplicon.” Nucleic acids can also be amplified in an isothermal reaction involving a reverse transcriptase, which copies   RNA →DNA, and a DNA-dependent RNA polymerase, which transcribes DNA→RNA. Isothermal amplification does not generate double-stranded DNA, and it is mainly used for copying RNA. Ligase-based methods, including the so-called Ligase Chain Reaction (LCR), can be also used for specific DNA or RNA amplification. A fourth general method for nucleic acid amplification involves   cloning  the selected DNA molecule into bacterial or eukaryotic cells, allowing them to reproduce, and collecting the amplified DNA.

10. Importance of DNA Amplification DNA  copies produced through PCR  amplification  can be used in a large number of medical and forensic applications. It can likewise be used in the identification and detection of infectious diseases and for a wide variety of research purposes in the field of molecular genetics. Genetic testing.  A single molecule of DNA is so small that it lies beyond the limits of detection of most, if not all, assays. Even with sensitive radioisotopes, it would be difficult if not impossible to detect a single molecule of DNA. The reason PCR was so important and revolutionary to molecular biology is that one cold begin with a single molecule of starting material and amplify it to an amount suitable for further analysis, including but not limited to:

11. Polymerase Chain Reaction(PCR) Polymerase chain reaction  ( PCR ) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. It is a technique used to make numerous copies of a specific segment of  DNA  quickly and accurately. The polymerase  chain reaction  enables investigators to obtain the large quantities of DNA that are required for various experiments and procedures in  molecular biology ,  forensic analysis ,  evolutionary  biology, and medical diagnostics.

12. The PCR technique is based on the natural processes a cell uses to replicate a new DNA strand. Only a few biological ingredients are needed for PCR. The  integral  component is the  template DNA —i.e., the DNA that contains the region to be copied, such as a  gene . As little as one DNA  molecule  can serve as a template. The only information needed for this fragment to be replicated is the sequence of two short regions of  nucleotides  (the subunits of DNA) at either end of the region of interest. These two short template sequences must be known so that two  primers —short stretches of nucleotides that correspond to the template sequences—can be synthesized. The primers bind, or anneal, to the template at their complementary sites and serve as the starting point for copying. DNA synthesis at one primer is directed toward the other, resulting in replication of the desired intervening sequence. Also needed are free nucleotides used to build the new DNA strands and a DNA polymerase, an  enzyme  that does the building by sequentially adding on free nucleotides according to the instructions of the template.

13. Steps of PCR PCR is a three-step process that is carried out in repeated cycles. The initial step is the Denaturation  , or separation, of the two strands of the DNA molecule. This is accomplished by heating the starting material to temperatures of about 95 °C (203 °F). Each strand is a template on which a new strand is built. In the second step the Reduction in temprature to about 55 °C (131 °F) so that the primers can anneal to the template. In the third step the Increase in temprature to about 72 °C (162 °F), and the DNA polymerase begins adding nucleotides onto the ends of the annealed primers. At the end of the cycle, which lasts about five minutes, the temperature is raised and the process begins again. The number of copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient amount of DNA

14. Importance of PCR The  Polymerase Chain Reaction  ( PCR ) is an  important  tool for many applications. For example, it can be used to amplify a sample of DNA when there isn't enough to analyze (e.g. a sample of DNA from a crime scene, archeological samples), as a method of identifying a gene of interest, or to test for disease. Polymerase chain reaction (PCR) is often considered as one of the most important scientific advances in the field of molecular biology. With this revolutionary yet inexpensive biochemical technology, it’s possible to generate millions of DNA copies from a single strand of DNA. As a result of this, PCR is considered to be one of the most indispensable techniques used in medical and biochemical research laboratories.

15. Applications of PCR Polymerase chain reaction, or molecular photocopying as it is lovingly called by some people, can be used in a variety of applications. DNA copies produced through PCR amplification can be used in a large number of medical and forensic applications. It can likewise be used in the identification and detection of infectious diseases and for a wide variety of research purposes in the field of molecular genetics. Medical Applications Genetic testing. PCR was first used to analyze the presence of genetic disease mutations. Tissue typing prior to organ transplantation. Formulation of individualized cancer therapy treatments.

16. Forensic Applications Genetic fingerprinting. PCR can be used to incriminate or rule out suspects in a crime investigation. Parental testing. PCR can be used to confirm the biological parents of an adopted child and/or identify the remains of an unidentified body. Infectious Disease Detection and Identification Detection of the Human Immunodeficiency Virus (HIV), one of the most difficult viruses to detect, and other disease organisms such as those that cause middle ear infection, tuberculosis and Lyme disease. Early detection of several forms of cancer including leukemia and lymphoma. Detection of viral DNA and virulent sub-types, including those that caused earlier epidemics.

17. Applications in Molecular Biology Research DNA sequencing, DNA cloning and gene expression. PCR can be used to produce huge amounts of pure DNA samples from a limited source. Production of hybridization probes for both northern and southern blot hybridization. Analysis of DNA from ancient sources.

18. Cell Culture Cell culture  is one of the major tools used in  cellular  and  molecular biology , providing excellent model systems for studying the normal physiology and biochemistry of  cells  (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the  cells , and mutagenesis and carcinogenesis. Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. 

19. Importance of Cell Culture in Molecular Genetics Cell culture  is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of  cells  (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the  cells  and mutagenesis and carcinogenesis Cell cultures are an extremely important tool for healthcare scientists. They provide a model system for physiology and biochemistry of selected cells to be studied. By examining their physiology their aging pathway can be studied and their biochemistry allows processes such as metabolic rate to be observed. The cells interaction with drugs could also be observed which proves a useful tool for drug screening programs, clinical trials and pharmaceutical companies. Whatever the purpose for using cell cultures, it is an extremely consistent and reliable process that has good reproducibility of results that can be obtained using a batch of clonal cells.

20. DNA Cloning in bacteria DNA cloning  is a  molecular  biology technique that makes many identical copies of a piece of  DNA , such as a  gene . ...  Bacteria  with the correct  plasmid  are used to make more  plasmid DNA  or, in some cases, induced to express the  gene  and make protein. Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.

21. One of the most  important  contributions of  DNA cloning  and genetic engineering to cell biology is that they have made it possible to produce any of the cell's proteins in nearly unlimited amounts. Large amounts of a desired protein are produced in living cells by using expression vectors  Uses of DNA cloning Biopharmaceuticals.  DNA cloning  can be  used  to make human proteins with biomedical  applications , such as the insulin mentioned above. ... Gene therapy. In some genetic disorders, patients lack the functional form of a particular gene. ... Gene analysis.

22. Gel Electrophoresis Gel electrophoresis is a method for separation and analysis of macromolecules and their fragments, based on their size and charge. Gel electrophoresis  is a technique used to separate DNA fragments according to their size. DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel. DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones. When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as  bands , each representing a group of same-sized DNA fragments.

23. What is a gel? As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gels for DNA separation are often made out of a polysaccharide called  agarose , which comes as dry, powdered flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores. At one end, the gel has pocket-like indentations called  wells , which are where the DNA samples will be placed: Before the DNA samples are added, the gel must be placed in a  gel box . One end of the box is hooked to a positive electrode, while the other end is hooked to a negative electrode. The main body of the box, where the gel is placed, is filled with a salt-containing buffer solution that can conduct current. Although you may not be able to see in the image above (thanks to my amazing artistic skills), the buffer fills the gel box to a level where it just barely covers the gel. The end of the gel with the wells is positioned towards the negative electrode. The end without wells (towards which the DNA fragments will migrate) is positioned towards the positive electrode.

24. DNA fingerprinting uses gel electrophoresis to distinguish between samples of the genetic material. The human DNA molecules are treated with enzymes that chop them at certain characteristic points, thereby reducing the DNA to a collection of more manageably sized pieces. The DNA fragments are loaded into a gel and placed in an electrical field, which electrophoretically sorts the DNA fragments into various bands. These bands can be colored with a radioactive dye to make them visible to imaging techniques

25. Applications of gel electrophoresis In the separation of DNA fragments for  DNA fingerprinting  to investigate crime scenes. To analyze results of polymerase chain reaction. To analyze genes associated with a particular illness. In DNA profiling for taxonomy studies to distinguish different species.

26. Isolation of DNA The free  DNA molecules  are subsequently  isolated  by one of several methods. ... A small pellet of  DNA  can be collected by centrifugation, and after removal of the ethanol, the  DNA  pellet can be dissolved in water (usually with a small amount of EDTA and a pH buffer) for the use in other reactions. The first isolation of DNA was done in 1869 by  Friedrich Miescher . [1]  Currently it is a routine procedure in  molecular biology  or  forensic  analyses. For the chemical method, there are many different kits used for extraction, and selecting the correct one will save time on kit optimization and extraction procedures.  PCR  sensitivity detection is considered to show the variation between the commercial kits.

27. DNA extraction is one of the most modern of the biological sciences. Scientists and doctors use DNA extraction to diagnose many medical conditions to genetically engineer both plants and animals. DNA extraction can also be used to gather evidence in a crime investigation. Application. The ability to extract DNA is of primary importance to studying the genetic causes of disease and for the development of diagnostics and drugs. It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment and for determining paternity.

28. That,s all for now! Thanks for tolerating! Any question?