Advanced Biotechnology Techniques Chapter 14 Learning Outcomes Discuss
Advanced Biotechnology Techniques Chapter 14
Learning Outcomes § § § Discuss the benefits and implications of knowing the DNA sequences for humans and other organisms Explain how DNA is sequenced using the Sanger Method and the recent improvements that have increased the efficiency of this process Describe some of the tools used in genomics and the advances made possible by them Discuss the field and potential of proteomics and the methods used for protein study Outline the important application of the growing biotechnology fields for pharmacogenetics, environmental and marine biotechnology, and bioterrorism/biodefense
14. 1 Advanced DNA Topics – DNA Sequencing DNA sequencing, polymerase chain reaction (PCR), microarray, and bioinformatics have provided so much data that researchers must design, conduct, and report the results of their experiments in ways that are different from those that were standard just a generation ago. DNA Sequencing DNA sequencing includes all the techniques used to determine the order of nucleotides (A, C, G, and T) in a DNA fragment.
Vocabulary • DNA sequencing – pertaining to all the techniques that lead to determining the order of nucleotides (A, C, G, and T) in a DNA fragment • Dideoxynucleotide sequencing – a sequencing method that uses dd. NTP and d. NTPs in a predictable way to produce synthesis fragments of varying length; also called the Sanger Method • Dideoxynucleotides – nucleotides that have an oxygen removed from carbon number 3, abbreviated dd. NTPs • BLAST – an acronym for Basic Local Alignment Search Tool, a program that allows researchers to compare biological sequences • Cycling sequence – a technique developed in the late 1990 s that allowed researchers to run synthesis reactions over and over on samples, increasing the amount of sequencing product and the speed of getting results • Human Genome Project – a collaborative 10 -year project completed in 2000, which aimed to sequence the entire DNA code for the human organism
14. 1 Review Questions 1. How is dd. NTP different from a “regular” d. NTP? 2. When preparing sequencing-reaction tubes, each of the four d. NTPs are added, but just one kind of dd. NTP. Which are used in the highest concentrations, the d. NTPs or the dd. NTPs, and why? 3. Where on the Internet may one go to compare DNA sequence data? 4. What additional instrument is required for cycle sequencing?
14. 2 Advanced DNA Studies – Genomics is the study of an organism’s genome, meaning all the genes and other noncoding sequences. RNA and Genomics There are several forms of RNA interacting with DNA and other RNA molecules to turn some genes on and off. RNA of Interest • • • RNAi si. RNA micro. RNA
Vocabulary • Genomics – the study of all the genes and DNA code of an organism • Bioinformatics – the use of computers and databases to analyze and relate large amounts of biological data • Shotgun cloning – a method of cloning used during the sequencing of the human genome that involves digesting DNA into 500 bp pieces, generating libraries from those fragments, and eventually sequencing the libraries • RNAi – abbreviation for RNA interference, a type of double-stranded RNA that is chopped in small pieces then engulfed by the cell, and binds to and interferes with the cells native RNA or DNA, blocking protein production • si. RNA – abbreviation for short-interfering RNA, a type of single-stranded RNA oligo (fragment) that is created by scientists to target a gene for silencing • micro. RNA – small pieces of RNA that are known to interrupt posttranscriptional RNA function by binding to m. RNA as soon as it is made • Northern blot – a process in which RNA fragments on a gel are transferred to a positively charged membrane (a blot) to be probed by labeled c. DNA
14. 2 Review Questions 1. How much of the human genome was sequenced using shotgun cloning? 2. Name a plant and an animal whose entire genome has been put on a Gene. Chip® microarray for the purpose of studying gene function and expression. 3. Micro. RNA, RNAi, and si. RNA all operate in a similar fashion. What do they do?
14. 3 Advanced Protein Studies A single gene can encode for as many as 50 different protein forms. X-Ray Crystallography Protein (x-ray) crystallography visualizes the positions of atoms in a molecule based on x-ray wave diffraction, and the constructive and destructive interference that results off the atoms in the sample.
Mass Spectrometry The mass spectrometer is an instrument that measures the masses and relative concentrations of atoms and molecules. Nuclear Magnetic Resonance Nuclear magnetic resonance measures the spin on nuclei (protons) of particular isotopes in a magnetic field.
ELISA is one of the most frequently used assays in protein manufacturing. Western Blots Running samples on acrylamide gels can provide a great deal of information about the proteins in a sample. Protein Arrays Protein arrays are a fusion of microarray and ELISA techniques, processed on a “protein chip. ”
Vocabulary • Proteomics – the study of how, when, and where proteins are used in cells • Proteome – all of an organism’s protein and protein-related material • NMR – abbreviation for nuclear magnetic resonance, a technique that measures the spin of nuclei (protons) of isotopes in a magnetic field to study physical, chemical, and biological properties of proteins, including their structure in aqueous (watery) solutions • Protein (x-ray) crystallography – a technique that uses x-ray wave diffraction patterns to visualize the positions of atoms in a protein molecule to reveal its three-dimensional structure • X-ray diffraction pattern – a pattern of light intensities that develops when an x-ray beam is passed through a mounted crystalline structure • Salting out – a technique for crystallizing proteins that involves precipitating a sample of pure protein using a stringent salt gradient of sodium chloride, ammonium sulfate, or some other salt • Protein arrays – a fusion of technologies, where protein samples bound on the glass slide (protein chip) are assessed using antibodies or other recognition material
14. 3 Review Questions 1. What is the technique called that uses an x-ray to study the structure of protein crystal? What is the picture called? 2. What causes proteins to crystallize out of solution? 3. What binds to what during an ELISA? How can a technician know that a protein is present during an ELISA? 4. What binds to what during a Western blot? How can a technician know that a protein is present during a blot?
14. 4 Other Advances and Applications of Biotechnology • • • Pharmacogenetics Environmental biotechnology Biodefense Pharmacogenetics – Personalized Medicines Since every person’s DNA is different, doctors will be able to design and modify drugs to better meet individual needs.
Environmental Biotech This is a vast field with many applications for monitoring and correcting the health of entire species, populations, communities, and ecosystems. Biodefense: Protection Against Bioterrorism Biodefense is the term for all of the methods used to protect people from exposure to biological agents.
Vocabulary • Pharmocogenetics – a branch of biotechnology that involves utilizing the genetic and protein codes to design or improve medications • Environmental biotechnology – a field of biotechnology whose applications include monitoring and correcting the health of populations, communities, and ecosystems • Marine biotechnology – the study and manipulation of marine organisms, their component molecules, cells, tissues, or organs • Bioterrorism – the use of biological agents to attack humans, plants, or animals • Biodefense – relating to all the methods used to protect a population from exposure to biological agents • Nanotechnology – pertaining to all technologies that operate on a nanometer scale
14. 4 Review Questions 1. What is the name of the process by which strategies are used to solve environmental problems, such as oil spills, soil erosion, or fertizier pollution? 2. What is the function of the Autonomous Pathogen Detection System? 3. What is the approximate size of the instruments and products of nanotechnology? 4. Give an example of a personalized medicine.
Questions and Comments?
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