Biotechnology and Recombinant DNA Chapter 9. Biotechnology The use of microbiological and...
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Transcript of Biotechnology and Recombinant DNA Chapter 9. Biotechnology The use of microbiological and...
Biotechnology The use of microbiological and biochemical
techniques to solve problems and produce product
Recombinant DNA techniques Methods used to manipulate DNA to intentionally
genetically alter organisms through genetic engineering
Often to give organisms more useful traits
Biotechnology and Recombinant DNA
Fundamental Tools of Biotechnology
Basic components of molecular biologist’s “toolkit” Restriction enzymes Gel electrophoresis DNA probes Primers
Restriction enzymes (Extracted from Bacteria) Naturally occurring enzymes that cut DNA into
fragments 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 file:///N:/Biology/Power%20Points/20_Lectures_PP
T/media/20_03RestrictionEnzymes_A.swf
Fundamental Tools of Biotechnology
Fundamental Tools of Biotechnology
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
DNA probes Used to locate nucleotide sequences in DNA
or RNA Probe is single-stranded piece of DNA tagged
with detectable marker Location can be easily determined
Probe will hybridize to complementary fragment of interest
Fundamental Tools of Biotechnology
Primers Single stranded DNA fragments that bind
sequences of DNA Used in in vitro DNA synthesis
Primer serves fragment for addition of DNA nucleotides
Fundamental Tools of Biotechnology
Applications of Genetic Engineering Genetically engineered
bacteria Genetic engineering
relies on DNA cloning Process of
producing copies of DNA
Cloned DNA generally combined with carrier molecule called cloning vector
Insures replication of target DNA
Genetically engineered organisms have variety of uses Protein production DNA production Researching gene function and regulation file:///N:/Biology/Power%20Points/20_Lectures
_PPT/media/20_04CloningAGene_A.swf
Applications of Genetic Engineering
Protein production Produce commercially important proteins
Pharmaceutical proteins Human insulin
Vaccines Hepatitis B vaccine
Commercially valuable proteins Chymosin An enzyme that catalyzes the coagulation
of milk used in the production of cheese
Applications of Genetic Engineering
DNA production Researches interested in acquiring available
sources of specific DNA fragments Fragments used for
DNA study Looking genomic characteristics
DNA vaccines Looking at injecting DNA of pathogen to produce
immune response
Applications of Genetic Engineering
Applications of Genetic Engineering
Researching gene function and regulation Function and regulation can
be more easily study in certain bacteria
E. coli used often due to established protocols
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
Genetically engineered eukaryotes Yeast serve as important eukaryotic model for gene
function and regulation Plant or animal that receive engineered gene termed
transgenic organism Examples of genetically altered plants include
Pest resistant plants Corn, cotton and potatoes
Herbicide resistant plants Soybeans, cotton and corn
Plants with improved nutrient value Rice
Plants as edible vaccines Bananas and potatoes
Applications of Genetic Engineering
Applications of Probe Technologies 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)
DNA microarray
Applications of Probe Technologies
Colony blotting Used to detect specific DNA
sequences in colonies grown in agar plates
Colonies are transferred in place on nylon membrane
Colony blots are used to determine which cells contain genes of interest
Applications of Probe Technologies
Southern blotting Uses probes to detect
DNA sequences in restriction fragments separated using gel electrophoresis
Application of Southern blotting is locating DNA sequences similar to ones being studied
Applications of Probe Technologies
Fluorescence in situ hybridization (FISH) Uses fluorescently labeled probes to detect
certain nucleotide sequences Detects sequences inside intact cells
Specimens are viewed using fluorescence microscopes
FISH can be used to identify specific properties of bacteria
Mycobacterium tuberculosis in sputum sample
Applications of Probe Technologies
DNA microarray technologies DNA arrays are solid
supports with fixed patterns of different single stranded DNA fragments attached
Enables researches to screen sample for numerous sequences simultaneously
Applications for DNA Sequencing Knowing DNA sequence of particular cell
helps identify genetic alterations Alterations that may result in disease
Sickle cell anemia Due to single base-pair change in a gene
Cystic fibrosis Caused by three base-pair deletion
Applications of Polymerase Chain Reaction Creates millions of
copies of given region of DNA in matter of hours Technique exploits
specificity of primers Allows for selective
replication of chosen regions
Termed target DNA 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
PCR Presentation
Techniques Used in Genetic Engineering
Obtaining DNA to be cloned Generally through cell lysis
Generating a recombinant molecule Restriction enzymes and ligases are used to
create a recombinant molecule Introducing recombinant molecule into new host
Host acts as an “incubator” for DNA replication DNA-mediated transformation often used to get
DNA into host
Techniques used in Probe Technologies
Probe technologies include Colony blotting Southern blotting FISH Microarray technology
Techniques in colony and Southern blotting Blotting steps transfer sample to nylon
membrane Probe is added
Probe hybridizes with complementary sequence Process is used to locate positions of
hybridized probe
Techniques used in Probe Technologies
Techniques used in FISH Sample preparation is critical
Methods used depend on type of organism Specimen is applied to glass slide Fluorescent label is applied and incubated
Incubation allows for hybridization Specimen is view with fluorescence
microscope
Techniques used in Probe Technologies
Techniques Used in DNA Sequencing
Dideoxychain termination Elements for termination reaction include
Single-stranded DNA template Primer that anneal to template DNA polymerase Each of the nucleotide bases
One of these bases is labeled with marker for detection Dideoxynucleotides
Like deoxynucleotide counterparts but lack 3’ OH Incorporation causes chain termination
Special gel electrophoresis used to separate DNA fragments by size
Techniques Used in DNA Sequencing
Automated DNA sequencing Most automated systems
use fluorescent dyes to detect newly synthesized DNA
Gel electrophoresis used to separate fragments into colored bands
Laser used to detect color differences
Order of color reflects nucleotide sequence
Techniques Used in Polymerase Chain Reaction
Starting with double stranded DNA molecule, process involves number of amplification cycles
PCR requires three step amplification cycle Step 1: double stranded DNA denatured by heat Step 2: primers anneal to complementary sequence of
target DNA and DNA synthesis occurs with heat stable DNA polymerase
Step 3: duplication of target DNA DNA is amplified exponentially