Chapter 20 Biotechnology. Focus of Chapter u An introduction to the methods and developments in: u...
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Transcript of Chapter 20 Biotechnology. Focus of Chapter u An introduction to the methods and developments in: u...
Focus of Chapter
An introduction to the methods and developments in: Recombinant DNA Genetic Engineering Biotechnology
Genetic Engineering
The direct manipulation of genes for practical purposes.
Ex: Using E. coli to produce human insulin.
Biotechnology
The use of living organisms or their components to perform practical tasks.
Ex: the use of bacteria to digest oil spills.
Restrictive Enzymes
Cut DNA at specific nucleotide sequences called “restriction sites”.
Used to "cut and splice" DNA.
Obtained from bacteria. Ex. EcoRI and Hind III
Plasmids
A small ring of independently replicating DNA separate from main chromosomes.
Found in Prokaryotes and yeast
Plasmids
Used extensively in Biotechnology and Recombinant DNA.
Serve as a “vehicle” for transporting genes.
Comment – other “vehicles” are used in other methods
Steps for Plasmid Use
1. Get the DNA for the trait.
2. Insert DNA into the plasmid.
3. Bacterial Transformation.
4. Identification of the new trait.
Insertion
Placing foreign DNA into a plasmid.
Open plasmid with enzymes to create “sticky ends”.
Splice the new DNA and plasmid together.
Identification
Screening the altered cells for the desired gene.
Ex: Antibiotic sensitivity or the expression of a “new” trait (color, glowing etc.).
Comment Gene can’t be above a certain
size (12 kb) or a plasmid won’t work.
mRNA must not need splicing to remove introns.
DNA Sources
1. Organism - use a section of their chromosome.
2. cDNA - Complementary DNA - created copy of DNA from the mRNA transcript to avoid introns. Uses reverse transcriptase.
Other Vehicles
BACs – Bacteria Artificial Chromosome – handle inserts of 100-300 kb
YACs – Yeast Artificial Chromosome – have a centromere and telomomere, handle inserts >300 kb
How Used
1. DNA is denatured to produce pieces.
2. Piece of complement DNA is added as a “probe”. The probe has been “labeled”.
3. Look for where the probe goes in the DNA sample. (DNA Microarray)
DNA Microarray
Used to separate pieces into wells
Well that is “marked” will hold the correct piece of DNA
PCR
Polymerase Chain Reaction Method for making many
copies of a specific segment of DNA.
Also called “DNA Amplification”.
PCR - Method
1. Separate strands by heating (denature the DNA).
2. Cool slightly.
3. Build new strand from primers and nucleotides.
4. Repeat.
Importance - PCR
Can amplify any DNA with as little as one original copy.
Very useful in a variety of techniques and tests.
Gel Electrophoresis
Used to separate mixtures of DNA or proteins according to size.
Smaller pieces will move further.
RFLP Analysis
Restriction Fragment Length Polymorphisms.
Method for detecting minor differences in DNA structure between individuals.
Common in DNA fingerprinting
Method
1. Digest DNA with restrictive enzymes.
2. Separate pieces by Gel Electrophoresis
3. Identify sequences with probes.
RFLP - Results
Patterns of DNA markers or DNA fingerprint
Markers are inherited and can show relationships (Pedigree studies).
Cloning of Organisms
Reproducing an organism by asexual means.
Commonly done in plants. Shows the concept of
“Totipotency” – that a single cell can develop into a new organism.
Cloning in Animals
Has been done by nuclear transplantation.
Examples – Dolly Many other vertebrates have
now been cloned.
Cloning steps
1. Somatic cell is taken from donor parent and nucleus is removed.
2. Unfertilized egg is taken from surrogate parent and the nucleus is removed and replaced with the somatic nucleus.
3. The egg is now grown in vitro until it is an embryo
Cloning continued
4. Embryo is placed in the womb of the surrogate mother.
5. Embryo develops. 6. The new clone is born. (Identical to
the donor mother)
Therapeutic cloning
Goal is to produce a group of certain cells not an organism.
Start with embryonic stem cells.
Known as stem cell research
Stem Cells
Stem Cell – an unspecialized cell that reproduces itself or differentiates into other cells.
Research
Trying to use stem cells to replace damaged cells or body parts.
If can use own stem cells, avoids tissue rejection problems.
Forensic Uses
DNA fingerprints for crime solving – used in every TV crime show
DNA identification records – standard for the military
Comments
Links suspect bodily fluids to the crime scene, but doesn’t prove they committed the crime.
Results take MUCH longer than on TV shows.
Analysis of old evidence is reversing some sentences.
Agricultural Uses
1. Animals Increased milk production Increased feed utilization Increased meat production
Agricultural Uses
2. Plants Herbicide resistance Retard spoilage of fruits Insect resistance – BT corn Nitrogen-Fixation ability
Genetically Modified Organism or GMO
Produced by direct genetic manipulation, not traditional breeding practices.
FDA just approved sale of GMO animal products for human consumption.
Bioethics concerns
Future Of DNA Technology
Cloning of higher animals. Stem Cells - growth of
replacement tissues and organs. Gene therapy to correct DNA
defects. ?
Summary Know the basics of some of
the DNA technology techniques.
Know: Bacterial transformation lab How Gel electrophoresis works Restriction enzymes