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46 Cards in this Set

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What is recombinant DNA technology?
Recombinant DNA technology is a set of molecular techniques for isolating, altering, and studying DNA segments. Also called genetic engineering.
What is biotechnology?
Biotechnology is the industry of using recombinant DNA technology to develop new products.
What do restriction enzymes do?
Restriction enzymes recognize and make double-stranded cuts in DNA at specific nucleotide sequences. They are produced naturally by bacteria and are used as defense against viruses.
Describe the sequences that are recognized by restriction enzymes.
Sequences that are recognized by restriction enzymes are 4-8 base pairs long, and most enzymes recognize sequences of 4 or 6 base pairs. Most sequences are palindromic, and type II enzymes only work with palindromes.
What are the three types of restriction enzymes?
There are three types of restriction enzymes: Type I, type II, and type III. The name of each enzyme beings with an abbreviation to signify its bacterial origin.
Describe type I and type III restriction enzymes.
Type I and III restriction enzymes cut DNA at sites outside of the recognition sequences.
Describe type II restriction enzymes.
Type II restriction enzymes recognize specific sequences and cut the DNA within the recognition sequence. Almost all molecular genetics work is done with the type II enzymes.
Describe the HindIII restriction enzyme example.
HindIII cuts the sugar-phosphate backbone of each strand at a specific point and makes fragments with short, single-stranded overhanging ends, called cohesive ends.
What are cohesive ends? When will DNA have cohesive ends?
Cohesive ends are also called sticky ends, because they are complementary to each other and so they can spontaneously pair to connect the fragments. Any two fragments cut by the same enzyme will have cohesive ends.
What is the relationship between the recognition sequence length and the number of times it appears in a chromosome?
There is a relationship between the length of the recognition sequence and the number of times that it is present in a genome. There are fewer long recognition sequences than short recognition sequences because there is a greater chance of a particular sequence if it is shorter.
Describe what gel electrophoresis does, and the basic steps.
Gel electrophoresis separates DNA molecules based on their size and electrical charge. A porous gel is made from agarose (a polysaccharide from seaweed), which is melted in a buffer solution and poured into a mold. Wells at one end hold the samples, and an electric current passes through the gel. The DNA fragments go to the positive end of the gel because of their negative phosphate groups, and they separate by size (smallest go furthest).
What is autoradiography?
Autoradiography is when radioactively labeled DNA is detected with a piece of X-ray film placed on top of the gel and electrophoresed. Radiation from the labeled DNA exposes the film like a camera.
What are probes, and what are they used for?
Probes are used to find desired DNA fragments in a large pool of DNA. The probe is a DNA or RNA molecule with a base sequence complementary to the one you are looking for, so the bases will pair with the one you want.
What is the process of Southern blotting, and what is it used for?
Southern blotting is a technique for transferring denatured, single-stranded DNA fragments from a gel to a permanent solid medium. After the fragments are moved, the membrane is put in a solution of a radioactive or chemically labeled probe, which binds to any DNA fragments on the membrane with complementary sequences. Then the membrane is washed and you are left only with the fragments you want.
Describe Northern blotting.
Northern blotting is when the probe can reveal the size of a particular mRNA molecule, its relative abundance, or the tissues where it is transcribed when RNA is moved from a gel to a solid support.
Describe Western blotting.
Western blotting is when proteins are moved from a gel to a membrane. Here, the probe is normally an antibody that is used to find the size of a particular protein and the pattern of its expression.
What is gene cloning?
Gene cloning is when you amplify a specific piece of DNA by placing the piece in a bacterial cell and letting it replicate the DNA.
What is a cloning vector?
A cloning vector is a stable, replicating DNA molecule that a foreign DNA piece can attach to for its introduction into the cell.
What does a good cloning vector need to have? (three things)
A good cloning vector needs three characteristics: (1) an origin of replication that makes sure the vector is replicated in the cell, (2) selectable markers, which let any cells with the vector be found or selected, and (3) one or more unique restriction sites where a DNA fragment can be inserted.
What are plasmids?
Plasmids are circular DNA molecules in bacteria, and they are common vectors for cloning DNA in bacteria.
What is the easiest way to put a gene into a plasmid vector?
The easiest way to put a gene into a plasmid vector is to cut the foreign DNA (containing the gene) and the plasmid with the same restriction enzyme, making cohesive ends so that the plasmid and the foreign DNA get mixed together.
What are linkers, and what do they do?
Linkers are used to create a restriction site when one isn’t available at the place where the DNA needs to be cut. Linkers are small, synthetic DNA pieces that contain one or more restriction sites.
What is transformation, and when is it used?
Transformation is the ability of bacterial cells to get DNA from the external environment, and it is used when a gene is put into a plasmid, so that the plasmid can be introduced into bacterial cells. Some cells do it naturally, others have to be induced chemically or physically.
What are selectable markers, where are they found, and what are they for?
Selectable markers are on plasmids, and they let the cells with recombinant plasmids be detected.
What is one type of selectable marker? Describe it.
One kind of selectable marker is the lacZ gene, which has a series of unique restriction sites where a DNA fragment can be inserted to be cloned.
What is the PCR, and what does it need to be done?
The polymerase chain reaction (PCR) lets DNA fragments be multiplied a billionfold in a few hours. The reaction is catalyzed by a DNA polymerase, and replication needs two things: (1) a single-stranded DNA template, so new DNA can be copied and (2), a primer with a 3’-OH group that new nucleotides can be added onto.
What happens to the amount of DNA in PCR with each replication, and what are the structure of the primers?
The amount of DNA in the PCR doubles with each replication. The primers are normally DNA fragments 17-25 nucleotides long, and they are complementary to sequences on the template.
How does the PCR begin?
PCR begins with a solution of the target DNA, DNA polymerase, all four dNTPs, primers, and the magnesium ions and other salts needed for the reaction. The chain reaction has three steps.
What is the first step of the PCR?
The first step of PCR is when the starting solution is heated to 90-100º C to break the hydrogen bonds between the strands, to get the single-stranded templates. It stays at that temperature for 1-2 minutes.
What is the second step of the PCR?
The second step of PCR is when the DNA solution is cooled quickly to 30-65º C for less than a minute, so the DNA strands can’t reconnect, but the primers can attach to the template strands.
What is the third and final step of the PCR? What happens at the end of this step?
The third step of PCR is when the solution is heated for less than a minute to 60-70º C, so DNA polymerase can make new DNA strands. At the end of this step, two new double-stranded DNA molecules are made for each original target DNA molecule.
Describe the bacteria Bacillus thuringiensis, and what is produces.
The bacteria Bacillus thuringiensis produces a protein (the Bt toxin) that kills insects. It is a good insecticide because it is specific, doesn’t hurt other animals, and breaks down quickly in the environment.
What did Vaek do with the Bt toxin?
In 1987, Vaek genetically engineered tobacco plants to show to Bt toxin. He used restriction enzymes to cleave the Bt gene sequences from the plasmids where they were grown into fragments of a different size, then attached them to a gene (neo) that gives resistance to the kanamycin antibiotic, which is toxic to plants and other eukaryotes to make a selectable marker for genes with the Bt gene.
What was done with the genetic constructs of Bt toxin?
The synthetic sequences (genetic constructs) were inserted into an expression vector with a promoter to make sure that the introduced sequences would be transcribed, and poly(A) consensus sequences to make sure the mRNAs would be properly translated.
What happened to the bacteria after the Bt toxin genetic constructs were inserted into the expression vectors?
Then the bacteria were transformed with the expression plasmids, and sequences on the vector recombined with sequences on the Ti plasmid and transferred the gene constructs to the Ti plasmid.
What did Vaek do to tobacco plants with the genetically altered bacteria? What is the result of the experiment?
Tobacco leaf discs were infected with this bacteria, which put the Ti plasmids into the plant cells. Then, whole tobacco plants were regenerated from the leaf discs and selected for kanamycin resistance. The plants were also pest resistant!
What are RFLPs?
Restriction fragment length polymorphisms (RFLPs) are a group of markers that are used in gene mapping. They are variations (polymorphisms) in the patterns of fragments made when DNA molecules are cut with the same restriction enzyme.
What are restriction fragment length polymorphisms used to do? What is their relationship with Huntington disease?
Restriction fragment length polymorphisms can be used to detect linkage. There is a close correspondence between RFLP allele inheritance and the presence of Huntington disease, which means that the genes that code for the RFLP and Huntington disease are closely linked.
What is DNA fingerprinting, and what does it use?
DNA fingerprinting is when DNA sequences are used to identify specific people. Since some genome parts are so variable, each person’s DNA is unique. Most DNA fingerprinting uses microsatellites, or short tandem repeats (STRs).
Describe the microsatellites (STRs, short tandem repeats).
Microsatellites (or STRs) are very short DNA sequences that are repeated in tandem and are found all over the human genome. People have different numbers of repeat copies. STRs are detected with PCR; more repeats = a longer amplified DNA segment.
What is a transgenic organism?
A transgenic organism is an organism that has been permanently changed by adding a DNA sequence to its genome, and the foreign DNA that it carries is called a transgene.
How are transgenic organisms created?
Mice and other mammal’s oocytes are big enough to have DNA injected directly into them. After sperm penetration, the fertilized mouse egg has two pronuclei: one from the sperm and one from the egg. The pronuclei later fuse to form the embryo nucleus.
What happens to the cloned, linear DNA for transgenic organisms? What is done with it?
Normally, a few hundred copies of cloned, linear DNA are injected into a pronucleus. In a few injected eggs, cloned DNA copies integrate randomly into one of the chromosomes through nonhomologous recombination. Then the embryos are implanted in a pseudopregnant female, a surrogate mother.
What happens to the eggs created to be transgenic organisms through nonhomologous recombination?
Only 10-30% of the eggs survive, and only a few of the survivors have a copy of the cloned DNA stably integrated into a chromosome. Mice with the cloned DNA in their chromosomes are mated to make a strain of mice homozygous for the foreign gene.
What are siRNAs, and what do they do?
siRNAs are small RNA molecules that combine with proteins to make the RNA-induced silencing complex (RISC), which pairs with complementary sequences on mRNA and either cleaves the mRNA or keeps it from being translated.
How can siRNAs be produced, and what do geneticists use them for?
siRNAs can be produced by cloning DNA sequences that correspond to the siRNAs between two strong promoters. Geneticists use siRNAs to turn off the expression of specific genes.