© life_edu Lecture 4 Some Techniques in Biotechnology Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us The Mechanics of DNA

Issues in Biotechnology: Biotechnology, Our Society and Our Future OnCampus Live BCH 190, MIC 190, AFS 190, NRS 190, PLS 190 OnLine BCH 190 A Sweeping General Survey on Life and Biotechnology A Public Access College Course The University of Rhode Island Kimberly Nelson Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us

© life_edu A Sweeping General Survey on Life and Biotechnology The University of Rhode Island Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us BCH 190 Section I. The Mechanics of Life and General Biotechnology

© life_edu 3. Atoms, Cells and the Flow of Life 4. Some Techniques in Biotechnology A Sweeping General Survey on Life and Biotechnology A Public Access College Course The University of Rhode Island Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us The Mechanics of DNA

© life_edu Lecture 4 Some Techniques in Biotechnology Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us The Mechanics of DNA

Tools of the Trade The eppendorf tube and the pipetman are the standard stock and trade in the daily work of a molecular biologist

Tools of the Trade The eppendorf tube and the pipetman are the standard stock and trade in the daily work of a molecular biologist “On the body of the traditional P-Series pipet it says, in relief, “Gilson.” Warren Gilson, who earned his MD in 1940 at the Univ. of Wisconsin, invented and patented the mechanical basis for the popular adjustable pipet (US Patent No. 3,827,305, 1974), Nearly 40 yrs. After the patent, the Pipetman continues to be manufactured in France in a factory started by Gilson’s colleague, Eugene Marteau D’Autry. Shortly before Gilson’s patent issued Gilson sold the marketing and sales rights to Ken Rainin President of Rainin Instrument, because, Gilson says, ‘He was a good salesman.’”

“Eppendorf tubes and Pipetman For the Gold Rush” Innovative technologies become biotech products

Separation Techniques: The need to separate the components of Life Precipitation/Dissolution Filters Centrifugation Affinity Blots Magnetics Electrophoresis Etc.

The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high as 2,000,000 G (approx km/s²). Ultracentrifuges find important uses in molecular biology, biochemistry and polymer science, including separation of cellular structures and molecules.

Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Gel Electrophoresis: the separation of molecules, DNA, RNA and proteins by charge and size

What is a Gel?

Agarose is a long chain of sugar molecules, a polymer, derived from algae used in electrophoresis to separate molecules Two types of gel: Agarose (horizontal type) Polyacrylamide (vertical type)

How are Gels Loaded and Run?

Applications of Gel Electrophoresis DNA Fingerprinting DNA Recombinant Technology Forensics The Human Genome Project

DNA carries a net negative charge; it is negatively charged because the phosphates (red circles) that form the sugar-phosphate backbone of a DNA molecule have a negative charge.

The gel matrix acts as a sieve for DNA molecules. Large molecules have difficulty getting through the holes in the matrix. Small molecules move easily through the holes. Because of this, large fragments will lag behind small fragments as DNA migrates through the gel.

As the separation process continues, the separation between the larger and smaller fragments increases.

Molecular weight markers are often electrophoresed with DNA. Molecular weight markers are usually a mixture of DNAs with known molecular weights. Molecular weight markers are used to estimate the sizes of DNA fragments in a DNA sample.

Issues in Biotechnology Gel electrophoresis is an important tool in molecular biology and biotechnology. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Gel electrophoresis allows for: (A) the separation of biological molecules, including DNA, RNA and proteins by their charge and size (B) all of the answers are correct (C) the identification of DNA markers now commonly used in forensics to implicate or exonerate persons accused of various crimes (D) the rapid visualization of the products of PCR (E) the acceleration of DNA into cells for genetic engineering purposes

The Techniques of Molecular Biotechnology Technology has created new Fields DNA detection Genomics DNA synthesisBioinformatics DNA sequencingPharmacogenomics DNA cloning Transgenics Expression cassette Computational constructionBiology RNA detection Population Genetics Protein detection Proteomics

The Techniques of Molecular Biotechnology Technology has created new Fields DNA detection Genomics DNA synthesisBioinformatics DNA sequencingPharmacogenomics DNA cloningTransgenics Expression cassette Computational constructionBiology RNA detection Population Genetics Protein detection Proteomics

DNA does not replicate spontaneously, but is facilitated by a group of proteins Interestingly, each of these proteins is coded for in DNA they also replicate Proteins Are Used to Copy DNA

Enzymes were discovered that cut DNA at specific sequences And subsequently, enzymes were discovered that paste DNA together The ability to cut and paste DNA allowed gene cloning

Plasmids are circular pieces of DNA found in some bacteria Many copies per cell Antibiotic resistance gene Plasmids can be cut and pasted back together Foreign genes can be inserted

How is a gene cloned? Foreign DNA (gene) is inserted into a plasmid that has a gene for antibiotic resistance The plasmid is introduced into a bacterial cell and grown on the antibiotic Only bacteria with the plasmid grow…the inserted gene is copied many times

PromoterCoding SequenceTerminator Gene Construction Cell specificity Developmental specificity Start transcription Protein coding sequence Stop transcription Message stability Gene constructs can be moved into plants and the gene is expressed driven by the promoter sequence

It is now possible to clone any gene from any organism and move it into any other organism

Gene transfer from one organism to another is not new Image of two species of bacteria transferring viral phage particles Bacteria transfer genes to other bacteria and plants Now in nature there is another organism capable of transferring DNA: we call that organism a human being

Tools and Techniques used in Biotechnology

No Walls The Clear bead at the center changes everything There are no edges to my being now I have heard it said that there is a window That opens from one mind to another But where there are no walls There is no need for a window, or fitting a latch. Rumi 1279 AD

20. A Pipetman is: (A) the new biomedical device made by tissue engineering and now used to treat the damaged blood vessels of heart attack victims (B) a radical group of bioengineered superheroes in the Hollywood movie GATTACCA (C) a molecular biology tool used in the lab to measure small volumes of liquid common in biotechnology (D) a new type of bio-engineered crop plants that are drought tolerant (E) a new surgical tool used in to extract cancer cells

21. An ultracentrifuge consists of a rotor that spins tubes containing materials and is: (A) a component on a new type of microscope to allow cell components to be easily visualized (B) the laboratory tool developed by Robert Hooke in the 1660s that he used to discover cells (C) a tool used by cell biologists for separating and comparing cell components based on size and density (D) a component on the recent Rover Mars mission used to look for life on other planets (E) used only to enrich uranium for nuclear warheads

22. Cloning a gene: (A) is a method that has been banned in Europe but widely used in the US (B) is a method used to copy a specific segment of DNA (C) is a method used to reproduce an entire new organism from a donor cell (D) is a science fiction idea that has not yet been accomplished or proven (E) is a good idea in theory, but has not yet been accomplished

23. The atomic number of carbon is 6. Its nucleus must contain: (A) 6 neutrons and 6 protons (B) 3 protons and 3 neutrons (C) 6 neutrons and no electrons (D) 6 protons and no electrons (E) 6 protons and 6 electrons

24. A covalent bond is formed when: (A) two non-polar molecules associate with each other in a polar environment (B) a positively charged particle is attracted to a negatively charged particle (C) one atom gives up electrons to another atom (D) two atoms share electrons (E) two polar molecules associate with each other in a non- polar environment

25. Enzymes are usually: (A) proteins (B) carbohydrates (C) helpful bacteria (D) only available in health food stores (E) none of these answers are correct

26. Changing one amino acid within a protein could change what about that protein? (A) the primary structure of the protein (B) the overall shape of the protein (C) the function of the protein itself (D) the sequence of amino acids specified in the DNA sequence called a gene (E) all of the above

27. A shortage of phosphorus would make it difficult for an organism to manufacture: (A) DNA (B) proteins (C) cellulose (D) fats (E) none of these answers are correct

28. Which of the following is not a chemical reaction? (A) sugar and oxygen combine to form carbon dioxide and water (B) sodium metal and chlorine gas unite to form sodium chloride (C) hydrogen gas combines with oxygen to form water (D) ice melts to form liquid water (E) none of these answers are correct

29. The chemical units of information in DNA are: (A) ATGC (B) UAGC (C) DAMN (D) RNAI (E) XYZQ

30. In DNA replication an (A) adenine always pairs with: (A) cytosine (C) (B) thymidine (T) (C) guanine (G) (D) tyrosine (E) monosodium glutamate

31. Observation of and wonder at the workings of nature are what initiate “why” and “how” type questions. Science is a system of: (A) relying on one’s best intuition, inspiration and perspiration to solve problems (B) advertising as a devious enterprise conceived by entrepreneurial western capitalists to make money from the ideas of inventive and often eccentric minds (C) making theories that fit certain beliefs about why and how things happen (D) hypothesis making about the mysteries of life (E) hypothesis making and testing to discern and validate observable facts

32. Water can absorb and store a large amount of heat while increasing only a few degrees in temperature. Why? (A) the heat must first be used to break the hydrogen bonds rather than raise the temperature (B) the heat must first be used to break the ionic bonds rather than raise the temperature (C) the heat must first be used to break the covalent bonds rather than raise the temperature (D) an increase in temperature causes an increase in adhesion of the water (E) an increase in temperature causes an increase in cohesion of the water

33. Water is split during photosynthesis is plants to yield what to compounds? (A) methane gas and carbon dioxide (B) oxygen and water (C) hydrogen and oxygen (D) sugars and proteins (E) RNA and DNA