Biotechnology (Lec. 7 + 8) Flashcards
Define biotechnology
the exploitation of biological processes for industrial and other purposes, especially the genetic manipulation of microorganisms for the production of antibiotics, hormones, etc.
Define recombinant DNA technology
A technology that uses enzymes to cut and paste together DNA sequences of interest
Define plasmid
A genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan
Define a vector
A self-replicating DNA molecule used to transport foreign DNA into a cell (plasmid or viral genome); typically engineered from naturally occurring bacterial/yeast plasmid or from viruses.
Define restriction enzymes
Also called restriction endonucleases; recognize specific sequences within a DNA molecule and then make a double-stranded cut
Outline RE cloning
A restriction enzyme cuts double-stranded DNA at its particular recognition sites. These cuts produce a DNA fragment with two sticky ends. When two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing. They’ll usually form either a linear molecule or a circular one. DNA ligase is used to unite the backbones, producing a molecule of rDNA.
Describe why restriction enzymes evolved and how they function in nature.
They evolved as a defense mechanism in bacteria to protect against invading viruses, by recognizing and cutting specific sequences within the viral DNA, effectively preventing the virus from replicating within the bacterium.
List the main properties of cloning vectors.
Should be a reasonably small/manageable DNA molecule; it should be relatively easy to move the vector from one organism to another; should be straightforward to generate and purify large amounts of vector DNA.
Usually have a selectable marker, ability to insert genes into the vector, and ability to detect the presence of an inserted gene in the vector (reporter gene)
Describe the ways of introducing recombinant DNA into bacterial cells
Transformation: cells take up DNA from the surrounding environment
Electroporation: electrical current forms pores in cell membranes to facilitate transformation
Protoplast fusion: removing cell walls from two bacteria to allow them to fuse
Transduction: bacteriophage vectors
Describe the ways of introducing recombinant DNA into eukaryotic cells
Transfection: cells take up DNA from environment
Electroporation: electrical current forms pores in cell membranes to facilitate transfection
Gene gun: DNA-coated particles
Microinjection: using a tiny micropipette
Transduction: viral vectors
Explain how antibiotic-resistant genes are used to select a desired clone
You can select and engineer a vector to include an antibiotic-resistant gene, which will then be introduced to host cells. Then, the cells will be cultured on a plate containing that antibiotic, and the cells that survive will have the antibiotic-resistant gene (and whatever other genes you put in the vector)
Explain how DNA probes are used to select a desired clone
DNA probes have sequences of complementary DNA to the target sequence
Explain how reporter genes (lacZ) are used to select a desired clone
lacZ encodes beta-galactosidase, which can break down lactose, ONPG, and X-gal. When ONPG is broken down, it turns yellow and soluble. When X-gal is broken down, it turns blue. So if you’re trying to create specific recombinant DNA, you can direct foreign DNA to be inserted in the middle of the lacZ gene, and if it’s successful then there won’t be any fun colors if you put it in a medium containing ONPG or X-gal.
Explain how colony hybridization is used to select a desired clone
Colony hybridization uses DNA probes that carry short segments of single-stranded DNA complementary to the desired gene in a cell
Interpret the results of a blue-white screen
Only cells that receive the plasmid (which has a gene for ampicillin resistance) will grow on the plate, which contains ampicillin. Bacteria with plasmid will produce beta-galactosidase and break down X-gal, so the colonies will be blue. In bacteria that received the recombinant plasmid, the lacZ gene will have been disrupted, so they won’t be able to break down X-gal, so colonies will be white.
Describe the activity of beta-galactosidase as it relates to ONPG and X-gal
Beta-galactosidase will break down ONPG into components, one of which is yellow and soluble. Beta-galactosidase will also break down X-gal, which creates the precursor to a blue dye, and the precursor is activated by oxygen.
Describe how a genomic library is made
An organism’s DNA is spliced into plasmid or phage vectors or introduced into bacteria, and at least one clone exists for every gene in the organism
Differentiate cDNA from synthetic DNA
cDNA is made from mRNA by reverse transcriptase. A cDNA library for eukaryotes does not have all the extra unnecessary DNA (like introns and stuff). Synthetic DNA is artificially designed and chemically synthesized.
Define genomic library
A collection of cloned DNA fragments that represent the entire genome of an organism
Define cDNA library
A collection of cloned DNA sequences that are complementary to an organism’s mRNA
Describe the steps in traditional PCR
Primers, nucleotides, and DNA polymerase are incubated at 94 degrees C for 1 minute to separate the DNA strands. Then they are incubated at 60 degrees C for 1 minute, which allows primers to attach to single-stranded DNA. Then they are incubated at 72 degrees C for 1 minute, which is when DNA polymerase copies the target DNA. Repeat.
Define denaturation
The unfolding or breaking up of a protein
Define annealing
When two single strands of DNA stick together to form a double helix
Define elongation
The process by which an existing RNA strand becomes longer by incorporating additional nucleotides (by RNA polymerase)
Define amplicon
A piece of DNA or RNA that has been amplified (often the product of PCR)
Define primer
A short nucleic acid sequence that provides a starting point for DNA synthesis
Describe the principle of nucleic acid hybridization
Single-stranded DNA or RNA strands bond to form a double-stranded molecule
Describe the steps in Southern blotting procedure. List a use.
- DNA containing the gene of interest is extracted from human cells and cut into fragments (called restriction fragment length polymorphisms, or RFLPs) by restriction enzymes.
- The fragments are separated according to size by gel electrophoresis. The bands of each size of DNA can be made visible by staining.
- The bands are transferred to a nitrocellulose filter by blotting. The solution passes through the gel and filter to the paper towels on top by capillary action.
- This produces a nitrocellulose filter with DNA fragments positioned exactly as on the gel.
- The filter is exposed to a labeled probe for a specific gene. The probe will hybridize with a short sequence present on the gene.
- The fragment containing the gene of interest is identified by a band on the filter
Can be used to identify a gene’s presence and/or abundance in a genome.
Describe the steps in DNA fingerprinting. List a use.
- DNA extraction and amplification (PCR)
- DNA digestion/splicing by restriction enzymes
- DNA separation through gel electrophoresis
- Data comparison and interpretation: compare the banding pattern of the samples to identify the individual
Most often used in forensic biology to identify someone.
Describe the steps in DNA microarray. List a use.
Short oligonucleotides are bound to a glass slide in a microscope array, and if complementary molecules are in the sample, they will bind and be visualized by a detector.
Can determine whether the DNA from an individual contains mutations.
Describe RNAi and how it is used in genetic manipulation
RNA interference aims to control gene expression by cloning sequences encoding siRNA (small interfering RNA) into a plasmid and transferring it into a cell
Define bioinformatics
understanding gene function via computer-assisted analysis of DNA sequences
Define proteomics
determining proteins expressed in a cell
Define metagenomics
the study of genetic material directly from environmental samples, or from a variety of different organisms in one ecosystem
Define reverse genetics
an approach to discovering gene function from a genetic sequence
List at least five applications of DNA technology
Gene therapy, forensic science, vaccines, to synthesize human insulin, paternity tests, diagnosis of genetic diseases
