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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

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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

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Agenda

• Background

• Restriction Endonucleases

• Gel Electrophoresis

• Polymerase Chain Reaction (PCR)

• Plasmids

• DNA Fingerprinting

• Applications

• Ethical Dilemmas

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• 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

What is Recombinant DNA Technology?

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

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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

• 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 (1 of 5)

Restriction Enzymes (2 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 (3 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 (4 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

Restriction Enzymes (5 of 5)

Gel Electrophoresis (1 of 2)

• Gel electrophoresis– 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)

• 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.

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PowerPowersourcesource

GelGel

Mixture of DNAMixture of DNAmolecules ofmolecules ofdifferent sizesdifferent sizes

LongerLongermoleculesmolecules

ShorterShortermoleculesmolecules

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

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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

• Genetically engineered organisms have a variety of uses– Protein production

– DNA production

– Researching gene function and regulation

Applications of Genetic Engineering

Applications of Protein Production• 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

• 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 DNA Production

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

• 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

Applications of Eukaryotic Genetic Engineering

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?

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Questions?