Introduction to Studying DNA

1. Introduction to Studying DNA Chapter 4

2. 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 • 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

3. 4.1 DNA Structure and Function The manipulation of genetic information, DNA and RNA codes, is at the center of most biotechnology research and development.

4. 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.

5. 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. Nitrogenous bases from each strand bond to each other in the center through H-bonds. The H-bonds are rather weak; therefore, the two strands of DNA separate easily in high temperatures.

6. Similarities in DNA Molecules Among Organisms • All DNA molecules are composed of four nucleic monomers • Adenosine deoxynucleotide (A) • Cytosine deoxynucleotide (C) • Guanosine deoxynucleotide (G) • 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 • Nucleotides in each strand are oriented in the opposite direction of the other strand • Nitrogenous bases • DNA undergoes semiconservative replication

7. 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.

8. 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

9. 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 – 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

10. 4.1 Review Questions • Describe the relationship between genes, mRNA, and proteins. • Name the four nitrogen-containing bases found in DNA molecules and identify how they create a base pair. • The strands on a DNA molecule are said to be “antiparallel.” What does antiparallel mean?> • During cell division, DNA molecules are replicated in a semiconservative manner. What happens to the original DNA molecule during semiconservative replication?

11. 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

12. Prokaryotic DNA Bacterial Operon. An operon contains the controlling elements that turn genetic expression ON and OFF.

13. Bacterial Cell Culture

14. Eukaryotic DNA Eukaryotic Gene. Eukaryotic genes have a promoter to which RNA polymerase binds, but they do not have an operator region.

15. 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 – refers to those 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

16. Vocabulary • Agar – 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 • 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 • Transcription factors – molecules that work to either turn on or off the transcription eukaryotic genes • Histones – nuclear proteins that bind to chromosomal DNA and condense it into highly packed coils • Nonpathogenic – not known to cause disease • Bacteriophages – viruses that infect bacteria • Gene therapy – the process of treating a disease or disorder by replacing a dysfunctional gene with a functional one

17. 4.2 Review Questions • Plasmids are very important pieces of DNA. How do they differ from chromosomal DNA molecules? • Bacteria cell DNA is divided into operons. Describe an operon using the terms promoter, operator, and structural gene. • Describe the human genome by discussing the number and types of chromosomes, genes, and nucleotides. • What is gene therapy? Cite an examples of how it can be used.

18. 4.3 Isolating and Manipulating DNA • Identification of the molecule(s) • Isolation of the instructions (DNA sequence/genes) for the production of the molecule(s) • Manipulation of the DNA instructions • Harvesting of the molecule or product, testing it, and marketing it

19. Recombinant DNA Technology Methods to create new DNA molecules Site-Specific Mutagenesis 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

20. Vocabulary • Bioremediation – the use of bacteria or other organisms to restore environmental conditions • Site-specific mutagenesis – a technique that involves changing the genetic code of an organism (mutagenesis) in certain sections (site-specific)

21. 4.3 Review Questions • Genetic engineering by any method requires certain steps. Put the following steps in the correct order: • isolation of the instructions (DNA sequence/genes) • harvest of the molecule or product; then marketing • manipulation of the DNA instructions • identification of the molecule to be produced • What “naming” designation is used with recombinant products made through genetic engineering? • What is the smallest change in a DNA molecule that can occur after site-specific mutagenesis? What effect can this change have? • What gene has been the target of CF gene therapy? What does this gene normally do?

22. 4.4 Using Gel Electrophoresis to Study Gene Molecules Components of Gel Electrophoresis • Powdered agarose • Boiling buffer solution Agarose Gel Concentrates Most commonly used when separating pieces of DNA no smaller than 50 bp and no larger than 25,000 bp Gel Stains The gel is “run” until molecules of different sizes are thought to have completely separated.

23. Agarose Gel Tray. Gel trays differ depending on the manufacturer. Each has some method of sealing the ends so that liquid agarose can mold into a gel. Some gel trays, such as those made by Owl Separation Systems, make a seal with the box, so casting a gel is simple. Other trays require masking tape on the ends to make a mold. Still others, like the one shown here, have gates that screw into position: up for pouring the gel and down for running the gel. Molecules in a Gel Box. If negatively charged molecules are loaded into the wells and run on the gel, the smaller ones run faster and farther than the larger ones toward the positive electrode. This is because smaller molecules pass more easily through the tiny spaces of the gel network.

24. 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

25. 4.4 Review Questions • Agarose gels can be used to study what size of DNA fragments? • If agarose gel material is labeled 1%, what does the 1% refer to? • What causes molecules to be separated on an agarose gel? • Name two common DNA stains that are used to visualize DNA on agarose gels.

26. Questions and Comments?