Recombinant DNA Technology Chapter 8 Recombinant DNA Technology

The Role of Recombinant DNA technology in Biotechnology Intentionally modifying genomes of organisms for practical purposes Three goals Eliminate undesirable phenotypic traits Combine beneficial traits of two or more organisms Create organisms that synthesize products humans need

Overview of recombinant DNA technology Figure 8.1

The Tools of Recombinant DNA Technology Mutagens Physical and chemical agents that produce mutations Scientists utilize mutagens to Create changes in microbes’ genomes to change phenotypes Select for and culture cells with beneficial characteristics Mutated genes alone can be isolated

The Tools of Recombinant DNA Technology The Use of Reverse Transcriptase to Synthesize cDNA Isolated from retroviruses Uses RNA template to transcribe molecule of cDNA Easier to isolate mRNA molecule for desired protein first mRNA of eukaryotes has introns removed Allows cloning in prokaryotic cells

The Tools of Recombinant DNA Technology Synthetic Nucleic Acids Molecules of DNA and RNA produced in cell-free solutions Uses of synthetic nucleic acids Elucidating the genetic code Creating genes for specific proteins Synthesizing DNA and RNA probes to locate specific sequences of nucleotides Synthesizing antisense nucleic acid molecules

The Tools of Recombinant DNA Technology Restriction Enzymes Bacterial enzymes that cut DNA molecules only at restriction sites Categorized into two groups based on type of cut Cuts with sticky ends Cuts with blunt ends

Actions of restriction enzymes Figure 8.2

The Tools of Recombinant DNA Technology Animation: Recombinant DNA Technology

The Tools of Recombinant DNA Technology Vectors Nucleic acid molecules that deliver a gene into a cell Useful properties Small enough to manipulate in a lab Survive inside cells Contain recognizable genetic marker Ensure genetic expression of gene Include viral genomes, transposons, and plasmids

Producing a recombinant vector Figure 8.3

The Tools of Recombinant DNA Technology Gene Libraries A collection of bacterial or phage clones Each clone in library often contains one gene of an organism’s genome Library may contain all genes of a single chromosome Library may contain set of cDNA complementary to mRNA

Production of a gene library Figure 8.4

Techniques of Recombinant DNA Technology Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR) Large number of identical molecules of DNA produced in vitro Critical to amplify DNA in variety of situations Epidemiologists use to amplify genome of unknown pathogen Amplified DNA from Bacillus anthracis spores in 2001 to identify source of spores

Techniques of Recombinant DNA Technology Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR) Repetitive process consisting of three steps Denaturation Priming Extension Can be automated using a thermocycler

Techniques of Recombinant DNA Technology Animation: Polymerase Chain Reaction: Overview

Techniques of Recombinant DNA Technology Animation: Polymerase Chain Reaction: Components

Polymerase chain reaction (PCR) Figure 8.5a

Polymerase chain reaction (PCR) Figure 8.5b

Techniques of Recombinant DNA Technology Animation: PCR: The Process

Techniques of Recombinant DNA Technology Selecting a Clone of Recombinant Cells Must find clone containing DNA of interest Probes are used

Techniques of Recombinant DNA Technology Separating DNA Molecules: Gel Electrophoresis and the Southern Blot Gel electrophoresis Separates molecules based on electrical charge, size, and shape Allows scientists to isolate DNA of interest Negatively charged DNA drawn toward positive electrode Agarose makes up gel; acts as molecular sieve Smaller fragments migrate faster and farther than larger ones Determine size by comparing distance migrated to standards

Gel electrophoresis Figure 8.6

Techniques of Recombinant DNA Technology Separating DNA Molecules: Gel Electrophoresis and the Southern Blot Southern blot DNA transferred from gel to nitrocellulose membrane Probes used to localize DNA sequence of interest Northern blot – used to detect RNA Uses of Southern blots Genetic “fingerprinting” Diagnosis of infectious disease Demonstrate incidence and prevalence of organisms that cannot be cultured

The Southern blot technique Figure 8.7

Techniques of Recombinant DNA Technology DNA Microarrays Consist of molecules of immobilized single-stranded DNA Fluorescently labeled DNA washed over array will adhere only at locations where there are complementary DNA sequences Variety of scientific uses of DNA microarrays Monitoring gene expression Diagnosis of infection Identification of organisms in an environmental sample

DNA microarray Figure 8.8

Techniques of Recombinant DNA Technology Inserting DNA into Cells Goal of DNA technology is insertion of DNA into cell Natural methods Transformation Transduction Conjugation Artificial methods Electroporation Protoplast fusion Injection – gene gun and microinjection

Artificial methods of inserting DNA into cells Figure 8.9a/b

Artificial methods of inserting DNA into cells Figure 8.9c/d

Applications of Recombinant DNA Technology Genetic Mapping Locating genes on a nucleic acid molecule Provides useful facts concerning metabolism, growth characteristics, and relatedness to others Locating Genes Until 1970, genes identified by labor-intensive methods Simpler and universal methods now available Restriction fragmentation Fluorescent in situ hybridization (FISH)

Fluorescent in situ hybridization Figure 8.10

Automated DNA sequencing Figure 8.11

Sequencing Valuable for diagnostic identification as well Provides definitive pathogen identification (usually) depending on the accuracy of library, size of target- resolution is better

The steps for 16S rRNA (Sanger Sequencing) Template preparation (extraction) Amplification of region of interest (PCR) Cycle Sequencing (dye terminator)

The steps for 16S rRNA (Sanger Sequencing) Template preparation (extraction) Amplification of region of interest (PCR) Cycle Sequencing (dye terminator) Extension Product Purification Separation of the products (electrophoresis)

Common Sequencing Targets 16S Ribosomal RNA (rRNA) Heat shock proteins (hsp) Internal transcribed spacer (ITS) RNA polymerase subunit B (rpoB)

Applications of Recombinant DNA Technology Environmental Studies Most microorganisms have never been grown in a laboratory Scientists know them only by their DNA fingerprints Allowed identification of over 500 species of bacteria from human mouths Determined that methane-producing archaea are a problem in rice agriculture

Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications Protein synthesis Creation of synthetic peptides for cloning Vaccines Production of safer vaccines Subunit vaccines Introduce genes of pathogens into common fruits and vegetables Injecting humans with plasmid carrying gene from pathogen Humans synthesize pathogen’s proteins

Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications Genetic screening DNA microarrays used to screen individuals for inherited disease caused by mutations Can also identify pathogen’s DNA in blood or tissues DNA fingerprinting Identifying individuals or organisms by their unique DNA sequence

DNA fingerprinting Figure 8.12

Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications Gene therapy Missing or defective genes replaced with normal copies Some patients’ immune systems react negatively Medical diagnosis Patient specimens can be examined for presence of gene sequences unique to certain pathogens Xenotransplants Animal cells, tissues, or organs introduced into human body

Applications of Recombinant DNA Technology Agricultural Applications Production of transgenic organisms Recombinant plants and animals altered by addition of genes from other organisms

Applications of Recombinant DNA Technology Agricultural Applications Herbicide resistance Gene from Salmonella conveys resistance to glyphosate (Roundup) Farmers can kill weeds without killing crops Salt tolerance Scientists have removed gene for salt tolerance and inserted into tomato and canola plants Transgenic plants survive, produce fruit, and remove salt from soil

Applications of Recombinant DNA Technology Agricultural Applications Freeze resistance Crops sprayed with genetically modified bacteria can tolerate mild freezes Pest resistance Bt toxin Naturally occurring toxin only harmful to insects Organic farmers used to reduce insect damage to crops Gene for Bt toxin inserted into various crop plants Genes for Phytophthora resistance inserted into potato crops

Applications of Recombinant DNA Technology Agricultural Applications Improvements in nutritional value and yield Tomatoes allowed to ripen on vine and shelf life increased Gene for enzyme that breaks down pectin suppressed BGH allows cattle to gain weight more rapidly, Have meat with lower fat content and produce 10% more milk Gene for β-carotene (vitamin A precursor) inserted into rice Scientists considering transplanting genes coding for entire metabolic pathways

The Ethics and Safety of Recombinant DNA Technology Supremacist view – humans are of greater value than animals Long-term effects of transgenic manipulations are unknown Unforeseen problems arise from every new technology and procedure Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic

The Ethics and Safety of Recombinant DNA Technology Studies have not shown any risks to human health or environment Standards imposed on labs involved in recombinant DNA technology Can create biological weapons using same technology

Molecular Diagnostics

Pathogen Identification Accurate identification important Patient to patient: Isolate causing infection? colonizer/ contaminant choice and duration of antibiotic treatment appropriate infection control procedure Large Scale: Epidemiology antibiotic resistance patterns treatment plans and outcomes associated with a particular bacterium

Conventional Identification

Advantages Disadvantages Inexpensive Can be time consuming Allow id for most organisms Can be time consuming Ambiguous profile

Limitations require additional equipment and expertise? What to do when organism: does not fit biochemical pattern? slow growing? Unable to culture? require additional equipment and expertise? (ex. anaerobes and mycobacterium) Phenotypic characteristics are not static and can change due to stress or environment

Alternative to Conventional Method Identification.. ...molecular Alternative to Conventional Method Identification..

Examples of Molecular Tests DNA hybridization Nucleic acid amplification Sequencing Melt curve analysis Strand Displacement Amplification Single Stranded Conformation Polymorphism Ligase Chain Amplification