Introduction to Studying DNA PDF

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 ! Explain how molecular modeling is used to study the structure and ! function of DNA as well as other biomolecules. ! Diff...

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 ! Explain how molecular modeling is used to study the structure and ! function of DNA as well as other biomolecules. ! 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 ! 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  4.1 DNA Structure and Function Manipulation of genetic information is at the center of most biotechnology R&D. 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.”  DNA Structure: Nucleotide Chains in a Double Helix 3D Model of DNA Structure Two chains of nucleotides twisted into a helix, connected to each other, in the center, through hydrogen bonds (H-bonds). Each nucleotide contains a sugar molecule, a phosphate group, and a nitrogenous base. See the next slide.  DNA Structure: Nitrogenous Bases DNA Structure. The nucleotides in a nucleic acid such as DNA contain a sugar molecule, a phosphate group, and a nitrogenous base. Nucleotides from each strand bond to each other in the center through H-bonds. The nucleotides containing adenine bonds to thymine and guanine bonds to cytosine.  DNA Structure: Antiparallel Strands DNA Structure. The nucleotides in one chain of the helix face one direction, while those in the other strand face the other direction. This is called “antiparallel.” The H-bonds holding the antiparallel strands together are rather weak; therefore, the two strands of DNA separate easily in high temperatures or in the presence of certain enzymes. This allows for DNA replication and mRNA transcription for protein synthesis  Similarities in DNA Molecules Among Organisms 1. All DNA molecules are composed of four nucleic monomers 1. Adenosine deoxynucleotide (A) 2. Cytosine deoxynucleotide (C) 3. Guanosine deoxynucleotide (G) 4. Thymine deoxynucleotide (T) 2. Virtually all DNA molecules form a double helix 3. The amount of adenine equals the amount of thymine The amount of guanine equals the amount of cytosine 4. Nucleotides in each strand are oriented in the opposite direction of the other strand 5. Nitrogenous bases 6. DNA undergoes semiconservative replication  DNA 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 in the base pairs of the DNA strands The number and type of genes and noncoding regions The shape of the DNA strands A single, circular DNA molecule is found in bacteria cells. ~40,000X  Section 4.1 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 of 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 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 involved 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 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  4.2 Sources of DNA In nature, DNA is made in cells. For sources of DNA, use cells in nature or grow cultures of cells in the lab. Structure of a Bacterium Prokaryotic DNA Bacterial Operon. An operon contains the controlling elements that turn genetic expression ON and OFF.  Bacterial Cell Culture To grow bacteria cells in the laboratory, a scientist must provide an environment, or medium, that the cells “like.” Some bacteria grow well in a liquid medium (broth). Some bacteria prefer a solid medium, called agar. Some will grow well on or in either.  Eukaryotic DNA 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. Mammalian Cell Culture Growing mammalian cells in culture is more challenging than growing bacterial cells – more nutrients, etc. Mammalian cells are grown in a broth culture  Viral DNA Viruses do not have cellular structure. Viruses are tiny, from 25 to 250 nm. They are collections of protein and nucleic acid molecules (DNA or RNA) and become active once they are within a suitable cell. Viruses are classified according to the type of cell they attack: Bacterial (bacteriophages) Plant Animal  Section 4.2 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 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 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  Section 4.2 Vocabulary Agar – a solid media used for growing bacteria, fungi, plant, or other cells Broth – a liquid media used for growing 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 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 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  4.3 Isolating and Manipulating DNA “Genetic engineering” is used to describe virtually all directed modifications of the DNA code of an organism. Scientists attempt to alter the genetic code to alter protein production. 1.Identify a target molecule(s) 2.Isolate the instructions (DNA sequence/genes) for the production of the molecule(s) 3.Manipulate the DNA instructions 4.Harvesting of the molecule or product, testing it, and marketing it Recombinant DNA Technology Methods to create new DNA molecules Site-Specific Mutagenesis Site-specific mutagenesis – a technique that involves changing the genetic code of an organism (mutagenesis) in certain sections (site-specific) Process of including changes (mutagenesis) in certain sections (site-specific) on a particular DNA code Gene Therapy Process of correcting faulty DNA codes that cause genetic diseases and disorders  4.4 Using Gel Electrophoresis to Study Gene Molecules Gel electrophoresis uses electricity to separate molecules, based on the size, shape, and charge, in a gel slab Components of Gel Electrophoresis Agarose dissolved in electrophoresis buffer Electrophoresis gel box Power supply Visualization system Hot agarose solution is poured into a gel tray. A comb is added to form sample wells as the agarose cools and solidifies. Wells are formed at one end so that sample molecules can move across the gel and separate based on their characteristics.  Gel Box with Gel and Buffer For the gel box to conduct electricity and establish an electric field with a positive end (red wire) and a negative end (black wire), 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. Agarose gel electrophoresis is best used when separating pieces of DNA no smaller than 50 bp and no larger than 25,000 bp.  Agarose Gel Concentrations Agarose gels are made with concentrations ranging from 0.6% to 3% agarose in buffer. The concentration of a gel is of critical importance. The more agarose molecules in solution, the more strands intertwine to make the “strainer.” If the concentration is too high, larger molecules can’t move through the long, woven agarose molecules. The most common gel used for DNA fragment separation is 0.8% agarose which is good for most plasmid and restriction digestion fragments separate well. “Tighter” gels (2% or 3%) are used to separate smaller molecules, such as PCR products. Proteins and nucleic acids that are smaller than 50 bp are run on polyacrylamide gels in vertical gel boxes.  DNA Gel Stains Nucleic acids are colorless, so technician must “stain” gels to see the bands of separated molecules. There are a few DNA stains to choose from. Ethidium bromide (EtBr) is the most common DNA gel stain in most labs and considered the most sensitive to low concentrations of DNA. EtBr glows orange when it is mixed with DNA and exposed to UV. Since EtBr is a suspected mutagen, other stains such as LabSafe®, SYBR Safe, GelRed, GelGreen, or methylene blue have become popular. An ethidium bromide stained gel. Each of these stains interacts with the Each white band is thousands of DNA nucleic acid molecules in a way that they molecules of similar size. can be visualized by either UV or white light. Each has some pros and some cons These bands are PCR products that are in its use. 500 to 1000 bp in length.  DNA Samples on a Gel This gel represents what DNA samples from eukaryotic and prokaryotic sources might look like on a 0.8% agarose gel. Lane 1 DNA sizing standard (Lambda/HindIII) Lane 2 DNA sample about 7000 bp (plasmid) Lane 3 Plasmid restriction digestion Lane 4 Genomic Bacterial DNA. Lane 5 mRNA Lanes 6/7 Smears of DNA, assorted size pieces Lane 8 No nucleic acid. Lane 9 DNA strands, large, will not “load” Lane 10 No sample  Section 4.4 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 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 High through-put screening – the process of examining hundreds or thousands of samples for a particular activity