Download What is Recombinant DNA Technology?

Recombinant DNA
Technology
1
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
2
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
3
Agenda
•
•
•
•
•
•
•
•
Background
Restriction Endonucleases
Gel Electrophoresis
Polymerase Chain Reaction (PCR)
Plasmids
DNA Fingerprinting
Applications
Ethical Dilemmas
4
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
7
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 reassociated by heating and cooling; Once unwound, DNA can
be copied  PCR
8
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)
• 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)
Mixture of DNA
molecules of
different sizes
–
–
Longer
molecules
Power
source
Gel
+
+
Shorter
molecules
• 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/mai
nimages/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
blood
24
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
• 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
• 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?
37
Questions?
38