NOTES - CH 15 (and 14.3): DNA Technology (“Biotech”)

“TRADITIONAL” BIOTECH: -microorganisms to make wine / cheese BIOTECHNOLOGY: the use of living organisms or their components to do practical tasks “TRADITIONAL” BIOTECH: -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; 4) Cloning.

Recombinant DNA: Combining fragments of DNA from different sources; 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 firefly – it glows!

BIOLUMINESCENT CAT!

“Toolkit” for recombinant DNA technology involves: -restriction enzymes -DNA vectors -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 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)

Gene Splicing: GENE SPLICING = rejoining of DNA fragments after cutting with restriction enzymes – foreign DNA is recombined into a bacterial plasmid or viral DNA

VECTORS = carriers for moving DNA from 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 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 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; 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; plasmid Human gene

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 gene (makes whatever protein the gene encodes)!

Applications of DNA Technology: Recombinant bacteria in industry; Recombinant bacteria in medicine; Recombinant bacteria in agriculture; Transgenic animals; Transgenic plants.

Recombinant bacteria in industry: Bacteria that can:  break down pollutants;  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 diabetes;  the amino acid phenylalanine.

Recombinant bacteria in agriculture: Bacteria that:  protect crops against frost;  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.

Transgenic plants: Plants that are engineered to:  resist herbicides;  produce internal pesticides;  increase protein production.

Other DNA Technologies: Polymerase Chain Reaction (PCR); Human Genome Project; 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;

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 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 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; 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 Ethical issues Research funding Who can afford treatment?