notes - ch 15 (and 14.3): dna technology (“biotech”)...cloning a human gene (cont.)… 5) insert...
TRANSCRIPT
NOTES - CH 15 (and 14.3):
DNA Technology (“Biotech”)
VocabularyGenetic Engineering
Gene
Recombinant DNA
Transgenic
Restriction Enzymes
VectorsVectors
Plasmids
Cloning
Key ConceptsWhat is genetic engineering?
How do scientists use it?
♦♦♦♦BIOTECHNOLOGY: the use of living organisms or their components to do practical tasks
“TRADITIONAL” BIOTECH:
-microorganisms to make -microorganisms to make
wine / cheese
-selective breeding of
livestock
-production of antibiotics
**Practical goal of
biotech =
improvement of
human health and
food production
DNA Technologies:
1) Making a recombinant DNA molecule;
2) Gene therapy;
3) DNA fingerprinting;3) DNA fingerprinting;
4) Cloning.
Recombinant DNA:
• Combining fragments of DNA from different sources;
• Result: organisms with • Result: organisms with their DNA + foreign DNA…such organisms are known as: TRANSGENIC ORGANISMS.
Example of transgenic organism:
� Tobacco plant
that contains a
gene from a gene from a
firefly – it glows!
BIOLUMINESCENT CAT!
“Toolkit” for recombinant DNA technology involves:
-restriction enzymes
-DNA vectors
-host organisms-host organisms
RESTRICTION ENZYMES = enzymes that recognize and cut short, specific DNA sequences
Restriction Enzymes…
♦are used to cut out a specific DNA fragment from an organism’s genome;
♦ recognize sequences that are “palindromic” (the same letters “palindromic” (the same letters backward and forward);
♦ typically cut sequences in a “staggered” manner so that the two ends of the fragments are single-stranded;
Restriction Enzymes (cont.)…
♦ this creates “sticky ends” so that the DNA fragment from one organism will be complementary to the DNA fragment from another organism. (complementary base pairing)(complementary base pairing)
Gene Splicing:
• GENE SPLICING = rejoining of DNA fragments after cutting with restriction enzymes – foreign DNA is recombined into a bacterial plasmid or viral DNAa bacterial plasmid or viral DNA
VECTORS = carriers for moving DNAfrom test tubes back into cells
-bacterial plasmids (small, circular DNA molecules that replicate within bacterial cells)
-viruses
HOST ORGANISMS:
bacteria are commonly
used as hosts in genetic
engineering because:
� bacterial cells are simple, and grow
quickly, replicating and expressing any
foreign genes they carry.
Gene Cloning:
• Once the foreign DNA has been transferred into the host bacterial cell, it replicates every time replicates every time the cell divides;
• CLONES = genetically identical copies of a gene
Gene Expression:
• In addition to copying the introduced foreign gene, bacterial cells will also EXPRESS the genes (make the protein the gene encodes!)
• EXAMPLE: if the gene for human insulin is • EXAMPLE: if the gene for human insulin is inserted into a bacterial
plasmid and then into a
host bacterial cell, that cell
will start to make
HUMAN INSULIN!
Steps Involved in
Cloning a Human Gene:
1) Isolate human gene to clone;
2) Isolate plasmid from bacterial cell;
plasmid
Human gene
3) Add a restriction enzyme to cut out human gene & add same R.E. to open up bacterial plasmid (creates complementary “sticky ends”);
4) Combine human gene with bacterial plasmid;
Cloning a Human Gene (cont.)…
5) Insert recombinant DNA plasmid back into bacterial cell;
6) As bacterial cell reproduces, it makes copies of the desired gene…and expresses that of the desired gene…and expresses that gene (makes whatever protein the gene encodes)!
Applications of DNA
Technology:
• Recombinant bacteria in industry;
• Recombinant bacteria in medicine;
• Recombinant bacteria in agriculture;• Recombinant bacteria in agriculture;
• Transgenic animals;
• Transgenic plants.
Recombinant bacteria in
industry:
• Bacteria that can:
� break down pollutants;
� degrade oil spills;� degrade oil spills;
� extract minerals from ores.
Recombinant bacteria in
medicine:
• Bacteria that have received human genes and produce:
� human growth hormone;
� insulin to treat � insulin to treat
diabetes;
� the amino acid
phenylalanine.
Recombinant bacteria in
agriculture:
• Bacteria that:
� protect crops against frost;
� produce natural fertilizers;� produce natural fertilizers;
� prevent crops from spoiling after harvest.
Transgenic animals:
• Engineer / produce animals with
human diseases so that they can be
studied in detail.studied in detail.
Transgenic plants:
• Plants that are engineered to:
� resist herbicides;
� produce internal pesticides;
� increase protein production.� increase protein production.
Other DNA Technologies:
• Polymerase Chain Reaction (PCR);
• Human Genome Project;
• Gel Electrophoresis;• Gel Electrophoresis;
• Gene Therapy;
• DNA Fingerprinting
The Polymerase Chain Reaction (PCR)
♦ allows any piece of DNA to be quickly copied many times in the lab;the lab;
PCR (continued)…
♦ BILLIONS of copies of DNA are produced
in just a few hours (enough to use for testing);
In 6 cycles of PCR:
cycle 1: 2 copies
cycle 2: 4 copiescycle 2: 4 copies
cycle 3: 8 copies
cycle 4: 16 copies
cycle 5: 32 copies
cycle 6: 64 copies
cycle 20: 1,048,576!!
Polymerase Chain Reaction (PCR)
♦PCR is highly specific; only a small sequence is amplified
♦ only tiny amounts of DNA are needed.
Starting materials for PCR:
• DNA to be copied
• Nucleotides (A,G,C,T)
• Primers• Primers
• DNA polymerase
Applications of PCR:
♦ analyze DNA from tiny amounts of
tissue or semen found at crime scene;
♦♦ analyze DNA from single embryonic cells for prenatal diagnosis;
♦ analyze DNA or viral genes from cells infected with difficult to detect viruses such as HIV;
♦ used extensively in Human Genome Project (14.3)
PCR works
like a
copying
machine for
DNA!
Analysis of Cloned DNA:Gel electrophoresis
♦ separates DNA molecules based on SIZE
♦ a mixture of DNA fragments will be sorted into bands, each consisting of DNA molecules of the same length
YOUR DNA
MY DNA
Steps Involved in DNA Fingerprinting:
1) Collect DNA from a sample;
2) Perform PCR if necessary to make more DNA;
3) Cut DNA apart using RE’s
**Junk DNA (introns) will be cut at different places for different people, therefore producing different size fragments
DNA Fingerprinting (cont.)…
4) Electrophoresis is used to separate DNA
pieces on a gel to create a banding pattern;
5) Photo of DNA gel is taken as evidence;5) Photo of DNA gel is taken as evidence;
6) Banding patterns can then be compared.
Sample
1
Sample
2
DNA_DetectivePC.exe
Gene Therapy:
• GENE THERAPY = the insertion of normal genes into human cells to correct genetic disorders
• Diseases treated include:
� cystic fibrosis
� SCID
(immune deficiency)
Biotech Today &
Tomorrow
• Experimental• Experimental
• Ethical issues
• Research funding
• Who can afford
treatment?