Biotechnology Biology III

Bellringer 1/10/12 What are the 4 bases in DNA? What is Transcription? What is Translation?

What is Biotechnology? Biotechnology: is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields of requiring bio products.

Selective Breeding Choose organisms with the desired traits and breed them, so the next generation also has those traits –Nearly all domesticated animals and crops –Luther Burbank ( ) developed >800 diff varieties of plants in his lifetime

Selective Breeding For a long time, humans have selected the best plants and animals to breed Why? Examples? Milk Cows – produced 4,997 lbs... of milk/year – produced 16,915 lbs.... of milk/year Increasing the frequency of desired alleles in a population is the essence of genetic technology

Hybridization The act or process of mating organisms of different varieties or species to create a hybrid. In plants – often results in better lines – hybrids are larger, stronger, etc In animals – hybrids produced may be weaker and sterile –Ex – wolf x dog ---- weak wolf-dog –Ex – horse x donkey ---- mule (sterile)

Inbreeding Breeding two organisms that are very similar to produce offspring with the desired traits. –Ex – dog breeds Risks – might bring together two individuals that carry bad recessive genes – many purebred dogs have genetic disorders that mutts don’t get.

Inbreeding Mating between closely related individuals Why? Done to make sure that breeds consistently exhibit a trait and to eliminate undesired trait –Creates purebred lines Can be bad also –Can bring out harmful, recessive alleles in a “family”

Increasing Variation Induce mutations – the ultimate source of genetic variations among a group of organisms –Mutagens used – radiation and chemicals –Some organisms are formed that have more desirable variations.

Producing new kinds of bacteria Can expose millions of bacteria at one time to radiation – increases chances of producing a successful mutant. –Ex – bacteria that can digest oil have been produced this way

Producing new kinds of plants: Drugs that prevent chromosomal separation in meiosis have been used to create plants that have more than two sets of chromosomes (2n). These are called polyploid plants. –Ex – bananas, citrus fruit, strawberries, many ornamental flowers Diploid corn Tetraploid corn

What is Genetic Engineering? Making changes in the genetic code of a living organism. - Transferring of DNA/genes from one organism to another - Also called recombinant DNA technology or gene splicing.

What is Genetic Engineering? Genetic engineering can take place: –Within a species (switching genes between humans) –Or between species (switching genes between humans and bacteria) Why is this possible?

What is Genetic Engineering? Gene: holds the genetic information to build and maintain an organism’s cells and pass genetic traits to offspring.

What is Genetic Engineering? Genome: the entirety of an organism’s heredity information.

How does genetic engineering take place?

Manipulating DNA – tools of the molecular biologist 1. DNA extraction – open the cells and separate DNA from all the other cell parts.

Steps to DNA Extraction 1.Break the cells open to expose DNA 2.Remove membrane lipids by adding detergent 3.Precipitate DNA with an alcohol — usually ethanol or isopropanol. –Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol- soluble salt.

2. Cutting DNA Sequences of DNA are isolated using restriction enzymes. Use restriction enzymes – each one cuts DNA at a specific sequence of nucleotides. (Usually 4-6 nucleotides) This will make different lengths of DNA

What is the role/function of restriction enzymes in bacteria?

Many enzyme cut in palindromes –Ex: a protein only cuts at AATT, it will cut the two fragments at different points - not across from each other (called sticky ends) Called sticky ends because they want to bond with things due to their “open” end Restriction Enzymes

These sticky ends are beneficial, because if the same enzyme is used in both organisms, they will have identical ends and will bond with each other.

Restriction Enzymes The cut ends (because they are complementary) can reattach or pair up with any other DNA fragment or gene cut by the same restriction enzyme.

Restriction Enzymes Restriction enzymes are used to cut or cleave the source DNA into fragments called: RFLP’s (Restriction Fragments Length Polymorphism) –Because the restriction enzyme’s recognition sequence is likely to occur many times within the source DNA, cutting will produce many fragments of different lengths. –Different RFLP’s may be made by using different restriction enzymes that recognize different DNA sequences.

Some Commonly Used Restriction Enzymes Eco RI 5'-G | AATTC Eco RV 5'-GAT | ATC Hin D III 5'-A | AGCTT Sac I 5'-GAGCT | C Sma I 5'-CCC | GGG Xma I 5'-C | CCGGG Bam HI I 5'-G | GATCC Pst I I 5'-CTGCA | G

Plasmids Plasmids: is a DNA molecule that is separate from and can replicate independently of the chromosomal DNA

Examples of the insertion of genes

Bellringer 1/19/11 What does restriction enzymes do? How are restriction enzymes important for genetic engineering? How has biotechnology affected you today? –(Give at least one example)

3. Separating DNA RFLP can be separated (Based on their size) by electrophoresis –Since a 3-billion base sequence of the 4 DNA nucleotides can produce more varied combinations than there are humans, each of us should have a unique DNA sequence.

Separating DNA – Gel Electrophoresis 1.Place fragments at one end of a porous gel – we use agarose gel 2.Apply an electric current – The DNA is negatively charged and will travel toward the positive end of the gel. 3.The larger pieces of DNA move slower, the smaller ones faster. 4.Used to compare genomes of different organisms or different individuals. 5.Also used to locate and identify one particular gene out of an individual’s genome.

Gel Electrophoresis gslc

Click here for animation about gel electrophoresis

Using the DNA Sequence Sequence can be read, studied, and changed. Techniques used to study DNA sequences: –Use DNA polymerase and the 4 DNA bases to produce a new DNA strand complementary to unknown strand – some of the bases are dyed. Dye-labeled strands are then separated using gel electrophoresis and the order of the bands tells the DNA sequence of the unknown strand.

Cutting and Pasting – make recombinant DNA (DNA from two different organisms). –Cut out the gene to be inserted, use same restriction enzyme to cut the receiving DNA strand, attach the two DNA strands

Making Copies Polymerase Chain Reaction (PCR) is used to make many copies of the same piece of DNA like a photocopy machine makes copies of papers. This is useful if there is only a very small sample of DNA available (as that found in a small blood drop at a crime scene)

Once the DNA of interest is isolated it is recombined with another organisms’ DNA Cell Transformation: A cell takes in DNA from outside the cell. –The external DNA becomes a component of the cell’s DNA 3. Recombinant DNA

Cell Transformation A cell takes in DNA from outside the cell and that DNA then becomes part of the cell’s DNA. Bacteria – place DNA in the solution that bacteria live in, and some of that DNA will be taken in by the bacteria cells.

Bacteria Transformation using Recombinant DNA Cut a gene with a restriction enzyme out of a human cell (ex – gene for insulin or growth hormone work well) Cut a bacterial plasmid using the same restriction enzyme (DNA ends will be complementary) Insert Human gene into bacterial plasmid Insert plasmid back into bacterial cell Bacteria will multiply, and all offspring will have that gene – these bacteria will then follow the directions of the human gene and make the protein coded for (insulin or human growth hormone)

Transforming Cells Use bacterial plasmid to insert desired gene into DNA Foreign DNA is first joined to a small, circular DNA molecule known as a plasmid. Plasmids are found naturally in some bacteria and have been very useful for DNA transfer.

Transforming Animal Cells –Directly inject DNA into the nucleus of an egg – it will become part of the chromosomes. Has been used to replace specific genes. Glowing mouse cells in embryos that were made from sperm given the gene for bioluminescence from jellyfish – now all the cells glow!

Making Recombinant DNA Step 1: To “recombine” or insert genes form one organism –Must first cut out the desired gene using the restriction enzyme –With the same restriction enzyme, cut out a segment of DNA from a plasmid or virus.

Making Recombinant DNA Step 2: Because the two different sources of DNA (human and bacteria) were cut with the same restriction enzyme, the “sticky ends will allow their DNA to recombine.

Making Recombinant DNA Step 3: Insert Human gene into bacterial plasmid –Insert plasmid back into bacterial cell

Making Recombinant DNA Step 4: Bacteria will multiply, and all offspring will have that gene –these bacteria will then follow the directions of the human gene and make the protein coded

Checking for recombinant cells Recombinant molecules must be separated from molecules consisting of just donor DNA or plasmid DNA. The experimenter designs the process so that the plasmid contains two genes that each enable a cell to grow in the presence of a different antibiotic drug.

Applications of Genetic Engineering Gene for luciferase was isolated from fireflies and inserted into tobacco plants – they glowed! Transgenic organisms – contain genes from other species A transgenic mouse, which carries a jellyfish gene, glows green under fluorescent light.

Transgenic Organisms Bacteria - Make human proteins like insulin Plants – 52% of soybeans, 25% of corn in US in year Some produce natural insecticide, some resist weed-killers, may soon be used to produce human antibodies; rice with vitamin A.

Animals – mice with immune systems like humans; farm animals that grow faster and larger with extra copies of growth hormone genes; animals with leaner meat; chickens resistant to bacterial infections.

Cloning Clone – member of a population of genetically identical cells produced from a single cell – Dolly cloned – 1 st mammal (sheep) cloned. She got arthritis several years earlier than most sheep Died in 2003