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Biotechnology Chapter Four Lecture- DNA
 

Biotechnology Chapter Four Lecture- DNA

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Biotechnology Chapter Four Lecture- DNA

Biotechnology Chapter Four Lecture- DNA

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    Biotechnology Chapter Four Lecture- DNA Biotechnology Chapter Four Lecture- DNA Presentation Transcript

    • Introduction to Studying DNA Chapter 4
    • Learning Outcomes    Describe the structure and function of DNA and explain the process by which it encodes for proteins Differentiate between eukaryotic and prokaryotic chromosomal structure and explain how this difference impacts gene regulation in the two cell types Differentiate between bacterial cultures grown in liquid and solid media and explain how to prepare each media type using sterile technique
    • Learning Outcomes    Discuss the characteristics of viruses and their importance in genetic engineering Explain the fundamental process of genetic engineering and give examples of the following applications: recombinant DNA technology, site-specific mutagenesis, and gene therapy Describe the process of gel electrophoresis and explain how the characteristics of molecules affect their migration through a gel
    • The manipulation of genetic information, DNA and RNA codes, is at the center of most biotechnology research and development. Section 4.1- DNA Structure and Function
    • The Central Dogma of Biology. Proteins are produced when genes on a DNA molecule are transcribed into mRNA, and mRNA is translated into the protein code. This is called “gene expression.” At any given moment, only a relatively small amount of DNA in a cell is being expressed.
    • DNA Structure The nucleotides in one chain of the helix face one direction, while those in the other strand face the other direction. Each nucleotide contains a sugar molecule, a phosphate group, and a nitrogenous base.
    • Phosphodiester bonds connect sugar molecules. The phosphate groups have a negative charge.
    • Nitrogenous bases are purines (two rings) or pyrimidines (single ring)
    • Nitrogenous bases from each strand bond to each other in the center through H-bonds. A with T (2 H-bonds) G with C (3 H-bonds) Always purine with pyramidine giving the double strand consistent width
    • The H-bonds are rather weak; therefore, the two strands of DNA separate easily in high temperatures. G/C rich DNA will separate at slightly higher temps due to having more H-bonds
    • Similarities in DNA Molecules Among Organisms All DNA molecules are composed of four nucleic monomers Adenosine deoxynucleotide (A), Cytosine deoxynucleotide (C), Guanosine deoxynucleotide (G), and Thymine deoxynucleotide (T) Virtually all DNA molecules form a double helix The amount of adenosine equals the amount of thymine The amount of guanosine equals the amount of cytosine due to base pairing
    • Similarities in DNA Molecules Among Organisms DNA is antiparallel- ✦nucleotides in each strand are oriented in opposite direction of the other strand ✦one strand is 3’ to 5’ the other is 5’ to 3’ 4.
    • Similarities in DNA Molecules Among Organisms DNA is antiparallel- ✦nucleotides in each strand are oriented in opposite direction of the other strand ✦one strand is 3’ to 5’ the other is 5’ to 3’ 4.
    • Similarities in DNA Molecules Among Organisms 10 base pairs / turn-The double helix has a regular shape which is recognized by other molecules. 4. DNA undergoes semiconservative replication
    • DNA Replication. DNA replicates in a semiconservative fashion in which one strand unzips and each side is copied. It is considered semiconservative since one copy of each parent strand is conserved in the next generation of DNA molecules.
    • Variations in DNA Molecules • The number of DNA strands in the cells of an organism • The length of the DNA strands • The number and type of genes and noncoding regions • The shape of the DNA strands
    • Vocabulary • Chromatin – nuclear DNA and proteins • Gene – a section of DNA on a chromosome that contains the genetic code of a protein • Nitrogenous base – an important component of nucleic acids (DNA and RNA), composed of one or two nitrogen-containing rings; forms the critical hydrogen bonds between opposing strands of a double helix • Base pair – the two nitrogenous bases that are connected by a hydrogen bond; for example, an adenosine bonded to a thymine or a gaunine bonded to a cytosine
    • Vocabulary • Phosphodiester bond – a bond that is responsible for polymerization of nucleic acids by linking sugars and phosphates of adjacent nucleotides • Hydrogen bond – a type of weak bond that involves the “sandwiching” of a hydrogen atom between two fluorine, nitrogen, or oxygen atoms; especially important in the structure of nucleic acids and proteins • Pyrimidine – a nitrogenous base composed of a single carbon ring; a component of DNA nucleotides
    • Vocabulary • Purine – a nitrogenous base composed of a double carbon ring; a component of DNA nucleotides • Antiparallel – a reference to the observation that strands on DNA double helix have their nucleotides oriented in the opposite direction to one another • Semiconservative replication – a form of replication in which each original strand of DNA acts as a template, or model, for building a new side; in this model one of each new copy goes into a newly forming daughter cell during cell division
    • 4.1 Review Questions 2. 1. Describe the relationship between genes, mRNA, and proteins. 3. 2. Name the four nitrogen-containing bases found in DNA molecules and identify how they create a base pair. 4. 3. The strands on a DNA molecule are said to be “antiparallel.” What does antiparallel mean? 5. 4. During cell division, DNA molecules are replicated in a semiconservative manner. What happens to the original DNA molecule during semiconservative replication?
    • 4.2 Sources of DNA In nature, DNA is made in cells. Mammalian Cell Culture • Growing mammalian cells in culture is more challenging than growing bacterial cells • Mammalian cells are grown in a broth culture Viral DNA Viruses are classified according to the type of cell they attack: Bacterial (bacteriophages) Plant Animal
    • 4.2 Sources of DNA In nature, DNA is made in cells. Bacterial Cell Culture Some bacteria prefer a liquid medium (nutrient broth), and some prefer a solid medium (nutrient agar).
    • 4.2 Sources of DNA In nature, DNA is made in cells. Bacterial Cell Culture Agar plates contain nutrient broth gelled with agar protein.
    • 4.2 Sources of DNA In nature, DNA is made in cells. Bacterial Cell Culture All media must be prepared under sterile conditions.
    • 4.2 Sources of DNA In nature, DNA is made in cells. Bacterial Cell Culture Introduction of cells must be done under sterile conditions to ensure purity.
    • 4.2 Sources of DNA In nature, DNA is made in cells. Bacterial Cell Culture An autoclave sterilizes equipment and solutions with high pressure and temp.
    • 4.2 Sources of DNA In nature, DNA is made in cells. Bacterial Cell Culture Sterile technique is an important lab skill for a microbiologist.
    • 4.2 Sources of DNA In nature, DNA is made in cells. Bacterial Cell Culture Sterile technique is doing something without contamination by unwanted organisms or their spores.
    • Bacterial genes In prokaryotes the DNA is floating in the cytoplasm.
    • Bacterial genes There is one large, twisted, circular chromosome that contains essential genes.
    • Bacterial genes There may be plasmids- small circular DNA molecules with non-essential but often advantageous genes
    • Bacterial genes Plasmids are transferred between bacteria in nature and in the lab allowing easy transfer of genes between prokaryotes.
    • Bacterial genes Plasmids are good vectors for gene recombination. A vector is a vehicle used to transfer genetic material.
    • Prokaryotic DNA regulation is simple Bacterial Operon An operon contains the controlling elements that turn genetic expression ON and OFF.
    • Prokaryotic DNA regulation is simple RNA polymerase attaches to the promoter.
    • Prokaryotic DNA regulation is simple Attachment of an inhibitor to the operator region will block production turning the gene off.
    • Prokaryotic DNA regulation is simple -the lac operon in E.coli
    • Genes must be transferred along with appropriate regulatory sequences.
    • Eukaryotic DNA Eukaryotes have multiple chromosomes
    • Eukaryotic DNA Each chromosome contains a single linear (very long) DNA molecule wrapped around histone proteins.
    • Eukaryotic DNA Tightly coiled chromosomes hide genes. Some unwinding from histones must occur for gene expression.
    • Eukaryotic DNA There is much spacer DNA that does not code for anything.
    • Eukaryotic DNA Eukaryotic Gene. Eukaryotic genes have a promoter to which RNA polymerase binds, but they do not have an operator region. Transcription factors may bind at enhancer regions and increase gene expression.
    • Eukaryotic DNA RNA polymerase moves down the DNA molecule until it reaches a structural gene which is transcribed until a termination sequence is reached.
    • Eukaryotic DNA The transcribed RNA molecule has introns (noncoding) removed and exons spliced together which leave the nucleus as mRNA.
    • Vocabulary • Medium – a suspension or gel that provides the nutrients (salts, sugars, growth factors, etc.) and the environment needed for cells to survive; plural is media • Lysis – the breakdown or rupture of cells • R plasmid – a type of plasmid that contains a gene for antibiotic resistance • Transformed – the cells that have taken foreign DNA and started expressing the genes on the newly acquired DNA •
    • Vocabulary • Vector – a piece of DNA that carries one or more genes into a cell; usually circular as in plasmid vectors • Operon – a section of prokaryotic DNA consisting of one or more genes and their controlling elements • RNA polymerase – an enzyme that catalyzes the synthesis of complementary RNA strands from a given DNA strand •
    • Vocabulary • Promoter – the region at the beginning of a gene where RNA polymerase binds; the promoter “promotes” the recruitment of RNA polymerase and other factors required for transcription • Operator – a region on an operon that can either turn on or off expression of a set of genes depending on the binding of a regulatory molecule • Beta-galactosidase – an enzyme that catalyzes the conversion of lactose into monosaccharides
    • Vocabulary • Agar – a solid media used for growing bacteria, fungi, plant, or other cells • Media preparation – the process of combining and sterilizing ingredients (salts, sugars, growth factors, pH indicators, etc.) of a particular medium • Autoclave – an instrument that creates high temperature and high pressure to sterilize equipment and media • Enhancer – a section of DNA that increases the expression of a gene • Silencer – a section of DNA that decreases the expression of a gene
    • Vocabulary • Transcription factors – molecules that work to either turn on or off the transcription eukaryotic genes • Intron – the region on a gene that is transcribed into an mRNA molecule but not expressed in a protein • Exon – the region of a gene that directly codes for a protein; it is the region of the gene that is expressed • Histones – the nuclear proteins that bind to chromosomal DNA and condense it into highly packed coils
    • Vocabulary • Nonpathogenic – not known to cause disease • Bacteriophages – the viruses that infect bacteria • Gene therapy – the process of treating a disease or disorder by replacing a dysfunctional gene with a functional one
    • . . . . 4.2 Review Questions 1. Plasmids are very important pieces of DNA. How do they differ from chromosomal DNA molecules? 2. Bacteria cell DNA is divided into operons. Describe an operon using the terms promoter, operator, and structural gene. 3. Describe the human genome by discussing the number and types of chromosomes, genes, and nucleotides. 4. What is gene therapy? Cite an examples of how it can be used.
    • 4.3 Isolating and Manipulating DNA Basic steps of genetic engineering 1. Identification of the molecule(s)What do you want to produce? 2. Isolation of the instructions (DNA sequence/genes) for the production of the molecule(s)
    • 4.3 Isolating and Manipulating DNA Basic steps of genetic engineering 3. Manipulation of the DNA instructions -change the DNA or -move it to a new organism 4. Harvest the molecule or product and test it To make a profit, the product must also be marketed.
    • Recombinant DNA Technology Recombinant DNA is made by combining different DNA molecules. Recombinant DNA and the proteins produced from it have an “r” before their name. rhInsulin = recombinant human insulin
    • Recombinant DNA Technology
    • Recombinant DNA Technology
    • Recombinant DNA Technology http://www.dnalc.org/view/15255-Producing-humaninsulin-using-recombinant-DNA-Walter-Gilbert.html http://www.youtube.com/watch? v=mhvARWPS1zM
    • Site-Specific Mutagenesis Process of inducing changes (mutagenesis) in certain sections (sitespecific) on a particular DNA code
    • Site-Specific Mutagenesis Random! Expose cells to chemicals, viruses, or radiation causing random mutations. Screen mutants for desirable traits.
    • Site-Specific Mutagenesis http://www.youtube.com/watch? v=DjGLUnJSRYs directed!! http://video.google.com/videoplay? docid=5044846172948251835
    • Gene Therapy Process of correcting faulty DNA codes that cause genetic diseases and disorders
    • Gene Therapy Process of correcting faulty DNA codes that cause genetic diseases and disorders Manipulation must occur in mature multicellular organism
    • Gene Therapy Process of correcting faulty DNA codes that cause genetic diseases and disorders Manipulation must occur in mature multicellular organism Viruses are used to insert functional genes.
    • http://www.youtube.com/watch? v=gl2miunHTRI&feature=related
    • Vocabulary • Site-specific mutagenesis – a technique that involves changing the genetic code of an organism (mutagenesis) in certain sections (site-specific)
    • 4.3 Review Questions 1. Genetic engineering by any method requires certain steps. Put the following steps in the correct order:
    • 4.3 Review Questions 1. Genetic engineering by any method requires certain steps. Put the following steps in the correct order: isolation of the DNA sequence
    • 4.3 Review Questions 1. Genetic engineering by any method requires certain steps. Put the following steps in the correct order: isolation of the DNA sequence harvest of the molecule or product
    • 4.3 Review Questions 1. Genetic engineering by any method requires certain steps. Put the following steps in the correct order: isolation of the DNA sequence harvest of the molecule or product manipulation of the DNA instructions
    • 4.3 Review Questions 1. Genetic engineering by any method requires certain steps. Put the following steps in the correct order: isolation of the DNA sequence harvest of the molecule or product manipulation of the DNA instructions identification of the molecule to be produced
    • 4.3 Review Questions 1. Genetic engineering by any method requires certain steps. Put the following steps in the correct order: isolation of the DNA sequence harvest of the molecule or product manipulation of the DNA instructions identification of the molecule to be produced 2. What “naming” designation is used with recombinant products made through genetic engineering?
    • 4.3 Review Questions 3. What is the smallest change in a DNA molecule that can occur after site-specific mutagenesis? What effect can this change have? 4. What gene has been the target of CF gene therapy? What does this gene normally do? CFTR gene = cystic fibrosis transmembrane conductance regulator
    • 4.4 Using Gel Electrophoresis to Study Gene Molecules
    • 4.4 Using Gel Electrophoresis to Study Gene Molecules DNA is negatively charged and moves towards the + end of an electric field.
    • 4.4 Using Gel Electrophoresis to Study Gene Molecules DNA is negatively charged and moves towards the + end of an electric field. Moving molecules are separated by size in a “gel”. Smaller pieces travel farther.
    • 4.4 Using Gel Electrophoresis to Study Gene Molecules DNA is negatively charged and moves towards the + end of an electric field. Moving molecules are separated by size in a “gel”. Smaller pieces travel farther. DNA is chopped up by restriction enzymes before loading in gel.
    • 4.4 Using Gel Electrophoresis to Study Gene Molecules agarose gel- Medium to large DNA pieces (500bp - 25,000bp) Poly-acrylamide (aka PAGE)- smaller stuff like protein, RNA, or small DNA
    • Components of Agarose Gel Electrophoresis Powdered agarose + boiling TRIS buffer solution is made to specified concentration
    • Components of Agarose Gel Electrophoresis Powdered agarose + boiling TRIS buffer solution is made to specified concentration 0.8% is appropriate for most restriction enzyme digested DNA
    • Components of Agarose Gel Electrophoresis Powdered agarose + boiling TRIS buffer solution is made to specified concentration 0.8% is appropriate for most restriction enzyme digested DNA 3% agarose gels would be much slower running
    • Components of Agarose Gel Electrophoresis A loading dye is used to visualize the movement of molecules.
    • Components of Agarose Gel Electrophoresis A set of DNA fragments of known size are also run for comparison.
    • Gel Stains Methylene blue makes the DNA visible.
    • Gel Stains Ethidium bromide (EtBr) makes DNA glow orange in UV light. More sensitive More hazardous
    • Gel trays differ depending on the manufacturer. Each has some method of sealing the ends so that liquid agarose can mold into a gel. Agarose Gel Tray.
    • For the gel box to conduct electricity, the solution in the gel box must contain ions. Sodium chloride (NaCl) solution can be used, but other salts, such as TRIS or lithium, dissolved in water (called a “running buffer”), are better for conducting electricity. Molecules in a Gel Box.
    • Vocabulary • Gel electrophoresis – a process that uses electricity to separate charged molecules, such as DNA fragments, RNA, and proteins, on a gel slab • Agarose – a carbohydrate from seaweed that is widely used as a medium for horizontal gel electrophoresis • Polyacrylamide – a polymer used as a gel material in vertical electrophoresis; used to separate smaller molecules, like proteins and very small pieces of DNA and RNA
    • Vocabulary • Ethidium bromide – a DNA stain (indicator); glows orange when it is mixed with DNA and exposed to UV light; abbreviated EtBr • • Methylene blue – a staining dye/indicator that interacts with nucleic acid molecules and proteins, turning them to a very dark blue color
    • 4.4 Review Questions 1. Agarose gels can be used to study what size of DNA fragments?
    • 4.4 Review Questions 1. Agarose gels can be used to study what size of DNA fragments? 2. If agarose gel material is labeled 1%, what does the 1% refer to?
    • 4.4 Review Questions 1. Agarose gels can be used to study what size of DNA fragments? 2. If agarose gel material is labeled 1%, what does the 1% refer to? 3. What causes molecules to be separated on an agarose gel?
    • 4.4 Review Questions 1. Agarose gels can be used to study what size of DNA fragments? 2. If agarose gel material is labeled 1%, what does the 1% refer to? 3. What causes molecules to be separated on an agarose gel? 4. Name two common DNA stains that are used to visualize DNA on agarose gels.
    • Questions and Comments?