-
Biotechnology
- the use of microorganisms, cells ro cell compoennts to make a product
- ex. foods, antibiotics, enzymes, proteins, vitamins
-
Genetic engineering and Recombinant DNA technolgoy
insertion of genes in an organism to produce desired protesin/enzymes or creat new cells that produce chemicals that an organism doesn't naturally make.
-
Selection
- the culturing of a naturally-occurring microbe that produces a desired product.
- Two types:
- 1. Artificial selection
- 2.Natural selection
-
Natural selection
survival of the fittest in biotechn.
-
Artificial selection
selectiion by humans of organisms with desirable characteristics.
-
Mutation
- mutagens cause mutations that might result in a microbe with a desirable trait.
- Two types:
- 1. Site-directed mutagenesis
- 2. Random selection
-
Mutation - Site-directed mutagenesis
changing a specific gene in DNA to change a protein
-
Mutation - random selection
select and culture microbe with the desired mutation.
-
Restriction enzymes
- cut specific sequences of DNA
- Naturally occur in some species of bacteria. ex. to protect against incoming DNA to cut it up before it cuases any damage.
- Methylated cytosins - tells restriction enzymes not to cut its own DNA
-
different Restriction enzymes
- cuts are staggered, producing "skicky ends" - become single strands
- if two fragments of DNA from different sources have been cut with the same restriction enzyme, the two fragments will have complimentroy sticky ends and can recombine.
- DNA ligase will then covalently link the sugar-phosphate backbones, producing a recombinant DNA molecule.
-
Vectors
- Transport DNA into the desired cell
- ex. Plasmids, transposons, and viruses
-
Importanc characterists of Vectors
- Must be self replicating
- Small
- Should resist destruction by the recipient cell
- a. circular vectors are highly resilient
- b. Viral DNA more likely to remain intact.
- Should carry a marker gene, which makes it easy to retrieve clones containing the vector.
-
Shuttle vectors
are plasmids capable of existing in several different species, and are used to move genes from one species to another.
-
Gene therapy and vectors
Vectors used to insert functional genes into human cells that have defective genes.
-
Polymerase Chain Reaction (PCR)
used to make multiple copies of a piece of DNA.
-
PCR is used to:
- Clone DNA for recombination
- Amplify DNA to detectable levles
- Sequence DNA - to determain order of letters
- Diangose genetic disease
- Detect pahtognes
-
Steps of PCR:
- 1.Original piece of DNA placed in a test tube with DNA primers, Individual DNA necluotides, DNA polymerease. Than test tube placed in a thermalcycler.
- 2.Incubated at 94 C for 1 min to seperate the strands of the original piece of DNA
- 3. Temp lowered to 60 C for 1 min. - DNA primers attach to the individual DNA strands
- 4. Temp increased to 72 C for 1 min - DNA polymerease synthesized a complimentory strand of DNA
- 5. Cycle is repeated many times, resulting in an exponential increase in the number of DNA pieces, all are identical to original DNA
- 6. DNA polymerase taken from the thermophilic bacterium "Thermus aquaticus" becuase of high temps.
-
Ways a recombinant DNA can be inserted into a cell
- Transformation
- Electroporation
- Protoplast Fusion
- Gene gun
- Microinjeciton
-
Transformation
cell treated with chemicals to make them "competent" or able to take up external DNA. Ex. CaCl2, than heat shocked
-
Electroporation
- applied an electrical current that forms small pores in the plasma membrane so the DNA can pass through.
- To take up DNA better by those cell that have cell walls they need to be converted to protoplasts. (G + doesn't have cell walls, so thye take up DNA beter)
-
Protoplasts fusion
Protoplasts put into a solution with added polyethylene glycol that increases the rate of fusion of two cells so that the 2 chromosomes can undergo natural recombination and become one cell.
-
Gene gun
pariticles of gold are coated with DNA and shot out of a gne gun witha burst of helium so that it penetrates plant cell walls - doen to eukaryotes - they can survive better.
-
Microinjection
a glass micropipette is used to inject DNA into a cell - humans
-
Gene library
are made up of pieces of an entire genome stored in bacterial plasmids or in phages.
-
Synthetic DNA
made by a DNA synthesis machine - only short pieces
-
Cloning the genes of Eukaryotes
- Complementary DNA (cDNA) is made from an mRNA template by the enzyme reverse transcriptase.
- The result is a DNA molecules without introns.
- This is used to obtain eukaryotic genes that can then be inserted into a prokaryotes.
-
Selecting a clone
- from a gene library that contains your gene of interests
- two ways:
- 1. Blue-white screening
- 2. Colony hybridization
-
Blue-white screening
- selects for clones that have any recombinant DNA
- Process:
-
Colony hybridization
- is specific: identifies the clone with your gene of interest.
- DNA probes are short sequences of single-stranded DNA that are compoimentary to the desired gene.
- a. DNA probes are synthesized in the labe
- b. Some DNA probes contain fluorescent dye
- c. Some DNA probes contain radiocative phosphorous
- DNA probes will bind with the gene of interest and serve as indicators
|
|