Trends in Biotechnology 110516 Constructing and Screening a DNA Library.
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Transcript of Trends in Biotechnology 110516 Constructing and Screening a DNA Library.
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Trends in Biotechnology
110516 Constructing and Screening a DNA Library
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Constructing and Screening a DNA Library.
DNA libraries help to map and sequence genomes, and are screened for the target DNA.
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Genomic Library.Contains DNA fragments that
represent an entire genome.
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Created by the following steps: a) Total nuclear DNA is isolated and cut with a restriction enzyme. b) A cloning vector is also cut with the same enzyme. c) The two DNAs are mixed in a test tube and placed into host cells. d) The host cells are selected for the recombinant DNA by antibiotics.
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e) Colonies (if not using a bacteriophage) or plaques (if using a bacteriophage) on bacterial plates represent successful transformation.f) A collection of colonies or plaques represents a library. g) Can calculate how many clones are needed to represent a genome.
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Fig. 3.15 The steps
involved in the
construction of a DNA library
(Genomic).
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cDNA Library.Made from mRNA, and shows only
genes expressed by a cell at a given time.
Reduces the amount of DNA to be cloned because all the genome is not being used.
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Made this way: a) Get mRNA from cells, use the enzyme reverse transcriptase to make one strand of DNA from the mRNA.b) Degrade mRNA with a ribonuclease (an enzyme that breaks down RNA) or an alkaline ( 알칼리의 ) solution. c) Makes the second DNA strand with DNA polymerase.d) Add double-stranded DNA pieces, called “DNA linkers,” to the new DNA, and put the recombinant DNA into a vector.
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The RNA that is used has already been processed and does not contain regulatory elements such as promoters. Fewer clones represent a library, making screening less labor-intensive.
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Fig. 3.16 The
synthesis of a cDNA.
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Fig. 3.16 The synthesis of a
cDNA.
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Screening Libraries.DNA inserts are found by a
screening process called “nucleic acid hybridization.”
A known DNA sequence is used as a probe to find the clones or plaques with the DNA sequence we want.
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Screening libraries are made this way (Figure 3.17): a) Bacterial colonies are transferred from a bacterial plate to a nylon or nitrocellulose membrane. b) Membranes are treated to lyse the cells.
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c) DNA on the membrane is denatured, and single-strand DNA probes that are labeled attach to the desired DNA. d) Unattached probe is washed off, and the label gives off light to expose photographic film.
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Fig. 3.17 Colony hybridization is used to identify bacterial cells that
have a specific recombinant plasmid.
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Fig. 3.18 Probe hybridization (cDNA or DNA) to signal-stranded DNA in cloned
DNA.
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Expression Libraries.1. Made with a cloning vector that contains the required regulatory elements for gene expression, such as the promoter region (Table 3.2). 2. Can insert into host cells to produce a protein or create a library.
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3. Useful for finding a clone with the gene or cDNA of interest. 4. DNA probes or antibodies (a process called “antibody binding”) can be used to find a DNA sequence. Antibodies can also be used to find proteins by Western blotting.
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