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25 Cards in this Set
- Front
- Back
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recombinant DNA
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a DNA molecule formed in the lab by joining together DNA sequences from different biological sources; used more loosely to refer to the technology that is utilized to create and study these hybrid molecules
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recombinant DNA molecules
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-a vector joined to a DNA fragment. (the fragments produced by restriction enzymes are joined to other DNA molecules that serve as vectors, or carrier molecules.); artificial laboratory creations that are not found in nature
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clone
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identical copy of a specific DNA molecule
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restriction enzyme
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proteins that are used to generate specific DNA fragments; these enzymes recognize and cut DNA molecules at specific nucleotide sequences
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restriction fragments
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DNA fragments cut by restriction enzymes in cloning; the size of restriction fragments is determined by the number of times a given restriction enzyme cuts the DNA
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recombinant vectors
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vectors carrying an inserted DNA fragment; an example of a recombinant DNA mlc, produced by joining DNA from two different sources
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pUC18
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a plasmid vector that is useful for several reasons: It is small (so it can carry relatively large DNA inserts), has an origin of replication and can produce up to 500 copies of inserted DNA fragments per cell, it has had a large number of restriction-enzyme recognition sequences engineered into them (conveniently clustered n one region called a polylinker sites), and are easily identified (for example, it carries a fragment of the bacterial lacZ gene as a selectable marker, and the polylinker is inserted into this fragment; expression of lacZ causes bacterial host cells carrying puC18 to produce blue colonies when grown on medium containing a compound known as Xgal. if a DNA fragment is inserted anywhere in the polylinker site, the lacZ gene is disrupted and becomes inactive. Thus, a bacterial cell carrying pUC18 with an inserted DNA fragment forms white colonies on Xgal medium, whereas colonies carrying pUC18 plasmids without inserted DNA fragments form blue colonies).
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polylinker
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a segment of DNA that has been engineered to contain multiple sites for restriction enzyme digestions. Polylinkers are usually found in engineered vectors such as plasmids.
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polymerase chain reaction (PCR)
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a rapid method of DNA cloning that extends the power of recombinant DNA research and in many cases eliminates the need to use host cells for cloning. By copying a specific DNA sequence through a series of in vitro reactions, PCR can amplify target DNA sequences that are initially present in very small quantities in a population of other DNA molecules. In practice, the PCR involves three steps: 1.) The DNA to be cloned is denatured into single strands by heating to 90-95 degrees Celsius 2.) Temp is lowered to an annealing temp between 50 and 70 degrees C, which causes the primers to bind to the denatured, single-stranded DNA (primers are synthetic oligonucleotides complementary to sequences flanking the target DNA; these primers [one complementary to the 5' end and one complementary to the 3' end of the target DNA] serve as starting points for synthesizing new DNA strands complementary to the target DNA, and 3.) a heat-stable form of DNA polymerase (such as Taq polymerase) is added to the reaction mixture, which extends the primers by adding nucleotides in the 5'-3' direction, making a double-stranded copy of the target DNA.
so 3 steps involved are: denaturing of double stranded DNA, primer annealing, and extension by polymerase; these steps make up a CycLe that is repeated depending on the number of new DNA strands you want [the number is doubled each time] |
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genomic library
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ideally, contains at least one copy of every sequence in an organism's genome; are constructed using host-cell cloning methods, since PCR-clone DNA fragments are relatively small. In making a genomic library, DNA is extracted from cells or tissues and cut with restriction enzymes, and the resulting fragments are inserted into vectors. Because the human genome is so large, the choice of vector is a primary consideration in making the library.
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pulsed-field gel electrophoresis
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a version of gel electrophoresis that separates very large DNA molecules; was used to isolate yeast chromosomes to be used in the construction of chromosome-specific libraries. A cloned library of yeast chromosome III produced in this way was the starting point for the Yeast Genome Project, in which the yeast genome was sequenced
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cDNA
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complementary DNA; DNA that is complementary to the nucleotide sequence of the mRNA; a cDNA library contains DNA copies made from mRNA molecules present in a cell population at a given time, and therefore represents the genes being expressed in the cells at the time the library was made; clones in a cDNA library are not the same as those in a genomic library; a cDNA library is prepared by isolating mRNA from a population of cells
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primer annealing
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the second step of PCR. the primers bind (stick) to the denatured, single-stranded DNA at complementary sequences flanking the target DNA
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restriction map
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one of the first steps in characterizing a DNA clone; establishes the number of, order of, and distances between restriction-enzyme cleavage sites along a cloned segment of DNA, thus providing information about the length of the cloned insert and the location of the restriction enzyme cleavage sites within the clone. Restriction maps for different cloned DNAs are usually different enough to serve as identity tags for those clones. The data the maps provide can be used to reclone fragments of a gene or compare its internal organization with that of other cloned sequences.
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southern blot
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detects hybridization between complementary nucleic acid molecules; can be used to identify which clones in a library contain a given DNA sequence (such as robosomal DNA, a beta-globin gene, etc.) and to characterize the size of the fragments, thus permitting a restriction map to be made. Can also be used to identify fragments carrying specific genes in genomic DNA digested with a restriction enzyme. Fragments of genomic clones isolated by Southern blots can in turn be isolated and recloned, providing a way to isolate parts of a gene. Has two components: separation of DNA fragments by gel electrophoresis and hybridization of the fragments using labeled probes. (Gel electrophoresis can be used to characterize the number of fragments produced by restriction digestion and to estimate their molecular weights; hybridization characterizes the DNA sequences present in the fragments) To make a Southern blot, DNA is cut into fragments with one or more restriction enzymes and the fragments are separated by gel electrophoresis. In preparation for hybridization, the DNA in the gel is denatured with alkaline treatment to form single-stranded fragments. The gels is then overlaid with a DNA-binding membrane (ex: nylon). Transfer of the DNA fragments to the membrane is accomplished by placing the membrane and gel on a wick (often a sponge) sitting in a buffer solution. Layers of paper towels or blotting paper are placed on top of the filter and held in place with a weight. Capillary action draws buffer up through the gel, transferring the DNA fragments from the gel to the membrane. The filter is placed in a heat-sealed bag with a labeled single-stragned DNA probe for hybridization. DNA fragments on the filter that are complementary to the probe's nucleotide sequence bind to the probe to form double-stranded hybrids. Excess probe is then washed away, and the hybridized fragments are visualized on a piece of film. In addtion to characterizing cloned DNAs, Southern blots can be used to create restriction maps of segments within and near a gene and to identify DNA fragments carrying all or parts of a single gene in a mixture of fragments. Southern blots also detect rearrangements, deletions, and duplications in genes associated with human genetic disorders and cancers by comparing the pattern of bands in normal cells to those from patients with disorders or cancer.
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probe
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any DNA or RNA sequence that has been labeled in some way and is complementary to some part of a cloned sequence present in the library; used to screen a library to recover clones of a specific gene
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X-gal
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an analog of lactose; a medium used to determine if a bacterial cell carrying a pUC18 has an inserted DNA fragment. If a DNA fragment is inserted anywhere in the polylinker site, the lacZ gene is disrupted and becomes inactive. Thus, a bacterial cell carrying a pUC18 with an inserted DNA fragment forms white colonies on Xgal medium, whereas colonies carrying pUC18 plasmids without inserted DNA fragments form blue colonies
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IPTG
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a compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon.
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Taq polymerase
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a heat-stable form of DNA polymerase used in the PCR; it extends the primers by adding nucleotides in the 5'-to-3' direction, making a double-stranded copy of the target DNA
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primer
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used in PCR; short DNA fragments containing complimentary segments to the target sequence
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origin of replication (ori)
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sites where DNA replication begins along the length of a chromosome
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hyperchromic shift
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the striking increase in absorbance of DNA
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satellite DNA
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usually consists of short sequences repeated many times in the genome; DNA that forms a minor band when genomic DNA is centrifuged in a cesium salt gradient
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molecular hybridization
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based on the denaturation and renaturation of nucleic acids
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dideoxynucleotide
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a nucelotide containing a deoxyribose sugar lacking a 3' hydroxyl group. it stops further chain elongation when incorporated into a growing polynucleotide and is used in the Sanger method of DNA sequencing.
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