Term
| 5 general procedures for the cloning of DNA from any organism |
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Definition
1. cutting target DNA at precise locations
2. selecting a small carrier molecule of DNA capable of self-replication
3. joining two DNA fragments covalently
4. moving recombinant DNA from the test tube to a host cell
5. selecting of identifying host cells that contain recombinant DNA |
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Term
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Definition
| the small carrier molecules of DNA capable of self-replication |
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Term
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Definition
| composite DNA molecules comprising covalently linked segments from two or more sources |
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Term
| recombinant DNA technology (genetic engineering) |
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Definition
| the methods used to accomplish cloning DNA from any organism & related tasks |
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Term
| restriction endonucleases |
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Definition
| recognize & cleave DNA at specific sequences to generate smaller fragments |
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Term
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Definition
| joins the DNA fragment to be cloned to a suitable cloning vector; links the DNA molecules together |
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Term
| restriction-modification system |
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Definition
| the restriction endonuclease & the corresponding methylase |
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Term
| type II restriction endonucleases |
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Definition
| simpler, require no ATP, & catalyze the hydrolytic cleavage of particular phosphodiester bonds in the DNA within the recognition sequence itself (first isolated by Hamilton Smith in 1970) |
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Term
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Definition
| unpaired strands; they can base-pair with each other or with complementary stick ends of other DNA fragments |
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Term
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Definition
| the ends with no unpaired bases left due to restriction endonucleases that cleave both strands of DNA at opposing phosphodiester bonds |
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Term
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Definition
| synthetic DNA fragments used to create new DNA sequences; inserted between the ends that are being ligated |
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Term
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Definition
| inserted DNA fragments with multiple recognition sequences for restriction endonucleases |
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Term
| 4 features of the E. coli plasmid "pBR322" that make it useful in almost all cloning vectors |
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Definition
1. the plasmid pBR322 has an origin of replication (ori) (a sequence where replication is initiated by cellular enzymes)
2. the plasmid contains genes that confer resistance to the antibiotics tetracycline & ampicillin, allowing the selection of cells that contain the intact plasmid or a recombinant version of the plasmid
3. several unique recognition sequences in pBR322 are targets for restriction endonucleases, providing sites where the plasmid can be cut to insert foreign DNA
4. the small size of the plasmid facilitates its entry into cells & the biochemical manipulation of the DNA |
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Term
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Definition
| the laboratory process in which small plasmids can be introduced into bacterial cells |
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Term
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Definition
| an approach; transiently renders the bacterial membrane permeable to large molecules |
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Term
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Definition
| either permit the growth of a cell or kill the cell under a defined set of conditions |
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Term
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Definition
| a gene encoding a protein that causes the cell to produce a colored or fluorescent molecule |
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Term
| bacterial artificial chromosomes (BACs) |
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Definition
| vectors once such large segments of cloned DNA have been added that they are large enough to be thought of as chromosomes |
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Term
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Definition
| plasmids that can be propagated in cells of two or more species |
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Term
| yeast artificial chromosomes (YACs) |
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Definition
| developed due to research on large genomes & the associated need for high-capacity cloning vectors |
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Term
| pulsed field gel electrophoresis |
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Definition
| how genomic fragments are separated; a variation of gel electrophoresis that segregates very large DNA segments |
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Term
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Definition
| cloning vectors with the transcription & translation signals needed for the regulated expression of a cloned gene |
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Term
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Definition
| ex) autographia californica multicapsid nucleoplyhedrovirus; used for protein expression |
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Term
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Definition
| large circular DNAs that include the entire baculovirus genome along with sequences that allow replication of the bacmid in E. coli |
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Term
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Definition
| when the recombinant bacmid is isolated & transfected into insect cells; when the DNA used for transformation includes viral sequences & leads to viral replication; is followed by recovery of the protein once the infection cycle is finished |
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Term
| site-directed mutagenesis |
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Definition
| how specific amino acids may be replaced individually |
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Term
| oligonucleotide-directed mutagenesis |
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Definition
| when suitably located restriction sites are not present; used to create a specific DNA sequence change |
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Term
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Definition
| the product of the fused gene created when parts of two different genes can be ligated to create new combinations |
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Term
| polymerase chain reaction (PCR) |
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Definition
| amplifies the number of copies of a DNA segment if the sequence of at least the end portion of a DNA segment of interest is known; the process was conceived by Kary Mullis in 1983 |
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Term
| reverse transcriptase PCR (RT-PCR) |
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Definition
| can be used to detect sequences derived from living cells as opposed to dead tissues |
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