Recombinant DNA Technology

Key Terms Biotechnology Recombinant DNA Restriction Enzymes Gel Electrophoresis Polymerase Chain Reaction (PCR) Plasmids DNA Fingerprinting Southern Blot DNA Microarray In situ Gene Therapy Transgenic Human Genome Project

Objectives Review the properties of DNA Explain what recombinant DNA technology is Understand what restriction enzymes are and how they help with recombinant DNA technology Describe the use of gel electrophoresis Explain how the PCR works Understand how plasmids are used with recombinant DNA technology Give examples of the current applications of recombinant DNA technology What ethical questions arise from human gene therapy

Agenda Background Restriction Endonucleases Gel Electrophoresis Polymerase Chain Reaction (PCR) Plasmids DNA Fingerprinting Applications Ethical Dilemmas

What is Recombinant DNA Technology? A technology that uses DNA molecules produced artificially and containing sequences from unrelated organisms to produce molecules and/or organisms with new properties. First developed in the mid 1970s Produced the Biotechnology Industry

Why Use Recombinant DNA Technology? To find practical applications to improve human health and molecule production Examples include: Making gene products using Genetic Engineering Uses in basic research Medical uses  diagnosis of disease Making vaccines/antibiotics and other pharmaceutical products Forensic uses of DNA such as DNA fingerprinting Agricultural uses such making transgenic plants Foods Vitamins Biodegradation

History 1953-Watson & Crick determine the structure of DNA 1970-first restriction endonuclease isolated 1973-Boyer & Cohen establish recombinant DNA technology 1976-DNA sequencing techniques developed 1980-U.S. Supreme Court rules that genetically modified micro-organisms can be patented 1981-first DNA synthesizers sold 1988-PCR method published 1990-Human genome project initiated 1996-Complete DNA sequence of a eukaryote (yeast) determined 1997-Nuclear cloning of a mammal (a sheep named Dolly) 2003-Human genome sequenced

Useful properties of DNA DNA sequences specify gene locations  Map genes Restriction endonucleases cut at specific nucleotides  Cut and Splice Nucleotides H bond with complementary nucleotides  Gene Probes DNA hybridization allows recognition of specific genes  DNA Fingerprinting The complementary strands of DNA can be separated and re-associated by heating and cooling; Once unwound, DNA can be copied  PCR

Tools of Recombinant DNA Technology Some of the basic components of molecular biologist’s “toolkit” include: Restriction enzymes Gel electrophoresis PCR Plasmids DNA Fingerprinting

Restriction Enzymes (1 of 5) Bacterial origin = enzymes that cleave foreign DNA Named after the organism from which they were derived EcoRI from Escherichia coli BamHI from Bacillus amyloliquefaciens Protect bacteria from bacteriophage infection Restricts viral replication Bacterium protects it’s own DNA by methylating those specific sequence motifs

Restriction Enzymes (2 of 5)

Restriction Enzymes (3 of 5) Cut in predictable and controllable manner Generates pieces of DNA called restriction fragments These fragments can be joined to new fragments Enzymes produce jagged cuts called sticky ends Ends anneal together to form new strand DNA ligase covalently joins fragments Over 2500 enzymes have been identified, recognizing ~200 distinct sequences 4–8 bases long Many are available commercially from biotechnology companies

Restriction Enzymes (4 of 5) Type I Cuts the DNA on both strands but at a non-specific location at varying distances from the particular sequence that is recognized by the restriction enzyme Therefore random/imprecise cuts Not very useful for rDNA applications Type II Cuts both strands of DNA within the particular sequence recognized by the restriction enzyme Used widely for molecular biology procedures DNA sequence = symmetrical

Restriction Enzymes (5 of 5) Reads the same in the 5’  3’ direction on both strands = Palindromic Sequence Some enzymes generate “blunt ends” (cut in middle) Others generate “sticky ends” (staggered cuts) H-bonding possible with complementary tails DNA ligase covalently links the two fragments together by forming phosphodiester bonds of the phosphate-sugar backbones

Gel Electrophoresis (1 of 2) Used to separate DNA fragments according to size DNA is put into wells in gel Gel subjected to current DNA moves through the gel Fragments are separated according to size Large fragments remain high in the gel Small fragments migrate lower Gel must be stained to view DNA Stained with ethidium bromide solution

Gel Electrophoresis (2 of 2) + – Power source Gel Mixture of DNA molecules of different sizes Longer molecules Shorter DNA is placed on a tray filled with an agarose gel through which an electric current runs causing the fragments to move through the gel. Segments separate by how far they move in the gel according to size.

Polymerase Chain Reaction (PCR) Used to Amplify a specific region of DNA Requires: DNA as template Cycles of heating and cooling Thermocycler (or water baths) Pool of free dNTPs Taq (or other heat-stable) DNA polymerase Primers - annealed at appropriate temperatures

Polymerase Chain Reaction (PCR) http://users.ugent.be/~avierstr/principles/pcrcopies.gif

Plasmids Small circle of bacterial DNA Foreign DNA inserted into plasmid Plasmid delivers DNA into another cell Cell expresses foreign DNA

Plasmids http://www.utpa.edu/faculty/materon/3401/mainimages/inserting.gif

Example of a plasmid + insert (DNA of interest)

DNA Fingerprinting Tandem Repeats Short regions of DNA that differ substantially among people Many sites in genome where tandem repeats occur Each person carries a unique combination of repeats

DNA Fingerprinting DNA is cut and then separated based on size of the DNA “Stained” and pattern of sizes is viewed Identify or rule out criminal suspects Identify bodies Determine paternity

DNA Fingerprinting can solve crimes Defendant’s blood Blood from defendant’s clothes Victim’s

Recombinant DNA Procedures 1. Get DNA and recombine it Restriction enzymes 2. Copy DNA Cloning PCR 3. Analyze DNA Sequencing Molecular Fingerprinting

Applications of Genetic Engineering Genetically engineered bacteria DNA cloning Copies of DNA Cloned DNA combines with a carrier molecule (vector) Insures replication of target gene

Applications of Genetic Engineering Genetically engineered organisms have a variety of uses Protein production DNA production Researching gene function and regulation

Applications of Protein Production Commercially important proteins Pharmaceutical Human insulin 1982, produced by bacteria First recombinant drug approved by the FDA Vaccines Hepatitis B vaccine Valuable proteins Chymosin - enzyme that catalyzes the coagulation of milk used in the production of cheese

Applications of DNA Production Providing researchers sources of specific DNA fragments for: DNA analysis genomic characteristics DNA vaccines injecting DNA of pathogen to produce immune response

Applications of Gene Function Researching gene function and regulation Can be more easily studied in certain bacteria E. coli Gene expression can be studied by gene fusion Joining gene being studied to reporter gene Reporter gene encodes observable trait Trait makes it possible to determine changes in gene Fluoresce

Applications of Eukaryotic Genetic Engineering Yeast are excellent eukaryotic models Plant/animal that receives engineered gene called transgenic Transgenic Plants: Pest resistant Corn, cotton and potatoes Herbicide resistant Soybeans, cotton and corn Improved nutrient value Rice Edible vaccines Bananas and potatoes

Application of DNA Probing Variety of technology employ DNA probes Colony blotting Southern blotting check for specific DNA in electrophoresis samples Fluorescence in situ hybridization (FISH) check for specific DNA sequences in whole chromosomes detects sequences inside intact cells DNA microarray/chips enables researches to screen sample for numerous sequences simultaneously

Applications of PCR Creates millions of copies of fragment of DNA in hours Technique exploits specificity of primers Allows for selective replication of chosen regions Large amounts of DNA can be produced from very small sample Care must be taken to prevent contamination with external source of target DNA Basis for false-positive test results Extremely useful in DNA forensics

Applications for DNA Sequencing Determining the DNA sequence of particular cell helps identify genetic alterations May result in disease Sickle cell anemia single base-pair change Cystic fibrosis three base-pair deletion

Applications for DNA Forensics PCR can recreate limited quantities of DNA DNA molecule is cut with restriction enzymes Separate the fragments via gel electrophoresis DNA forms bands corresponding to the bases (no two people have the same sequence of bases) in the gel which are unique for each individual.

Applications for Gene Therapy Human genome difficult to manipulate Viruses insert genes into cultured human cells Very difficult to get modified genes to work properly Retroviruses  Contain RNA that is injected into host cell along with enzymes. Reverse Transcriptase converts the RNA to DNA. Integrase inserts the DNA into the host genome Adenoviruses  Contains DNA that is put in the host nucleus and transcribed. SCID-X1: designed to cure “bubble babies” with immune system that don’t work

Ethical Dilemmas from Recombinant DNA Technology Eugenic human engineering Selecting for “desirable” human traits Creation of “designer” babies Who should decide what genetic traits can or should be altered? The perfect human? Says who?

Questions?