Www. Albia Dugger Miami Dade College Eldra Solomon Linda Berg Diana W. Martin Chapter 15 DNA Technology and Genomics

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  • www.cengage.com/biology/solomon Albia Dugger Miami Dade College Eldra Solomon Linda Berg Diana W. Martin Chapter 15 DNA Technology and Genomics
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  • Biotechnology Studies of DNA sequences reveal the organization of genes and the relationship between genes and their products Recombinant DNA technology allows researchers to splice together DNA from different organisms in the laboratory Molecular modification (genetic engineering) alters an organisms DNA to produce new genes with new traits Biotechnology includes all commercial or industrial uses of cells or organisms
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  • 15.1 DNA CLONING LEARNING OBJECTIVES: Explain how a typical restriction enzyme cuts DNA molecules and give examples of the ways in which these enzymes are used in recombinant DNA technology Distinguish among a genomic DNA library, a chromosome library, and a complementary DNA (cDNA) library; explain why one would clone the same eukaryotic gene from both a genomic DNA library and a cDNA library Explain how researchers use a DNA probe Describe how PCR amplifies DNA in vitro
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  • Recombinant DNA Technology Recombinant DNA technology began with genetic studies of viruses that infect bacteria (bacteriophages) Restriction enzymes from bacteria are used to cut DNA molecules in specific places a vector molecule transports the DNA fragment into a cell Bacteriophages and plasmids are two examples of vectors
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  • Recombinant DNA Technology (cont.) A plasmid with foreign DNA spliced into it (recombinant plasmid) is introduced into bacteria by transformation Once a plasmid enters a cell, it is replicated and distributed to daughter cells during cell division, producing many identical copies the foreign DNA is cloned
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  • Restriction Enzymes Restriction enzymes enable scientists to cut DNA from chromosomes into shorter fragments in a controlled way Each restriction enzyme cuts DNA at a specific DNA sequence (restriction site), such as 5-AAGCTT-3 Many restriction enzymes used for recombinant DNA studies cut palindromic sequences the base sequence reads the same as its complement, in the opposite direction such as 3-TTCGAA-5
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  • Sticky Ends Cutting both strands in a staggered fashion produces fragments with identical, complementary, single-stranded ends called sticky ends: 5-A AGCTT -3 3-TTCGA A-5 Sticky ends pair by hydrogen bonding with the complementary, single-stranded ends of other DNA molecules that have been cut with the same enzyme
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  • DNA Ligase Once the sticky ends of two molecules have been joined, they are treated with DNA ligase, an enzyme that covalently links the two DNA fragments to form a stable recombinant DNA molecule
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  • Cutting DNA with a Restriction Enzyme
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  • Fig. 15-1, p. 325 Plus HindIII restriction enzyme Sticky ends
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  • Recombinant DNA The DNA to be cloned and plasmid (vector) DNA are cut with the same restriction enzyme The two DNA samples are mixed, and complementary bases of the sticky ends are bonded The result is recombinant DNA
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  • Making Recombinant DNA
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  • Fig. 15-2, p. 325 Plasmid from a bacterium DNA of interest from another organism Clonable DNA fragment Recombinant DNA 1 2 3
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  • Fig. 15-2, p. 325 Plasmid from a bacterium DNA of interest from another organism 1 Recombinant DNA Clonable DNA fragment 2 3 Stepped Art
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  • Plasmids Plasmids used in recombinant DNA technology include features helpful in isolating and analyzing cloned DNA: One or more restriction sites Genes that let researchers select cells transformed by recombinant plasmids
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  • A Plasmid Vector
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  • Fig. 15-3a, p. 326 Aat I Xba I Hpa I Pvu II URA-3 Cla I Sal I Bam HI Sma I (a) This plasmid vector has many useful features. Researchers constructed it from DNA fragments they had isolated from plasmids, E. coli genes, and yeast genes. The two origins of replication, one for E. coli and one for yeast, Saccharomyces cerevisiae, let it replicate independently in either type of cell. Letters on the outer circle designate sites for restriction enzymes that cut the plasmid only at that position. Resistance genes for the antibiotics ampicillin and tetracycline and the yeast URA-3 gene are also shown. The URA-3 gene is useful when transforming yeast cells lacking an enzyme required for uracil synthesis. Cells that take up the plasmid grow on a uracil-deficient medium. E. coli origin of replication
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  • Plasmid in Bacterium
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  • Fig. 15-3b, p. 326 Bacterial chromosome Bacterium Plasmid (b) The relative sizes of a plasmid and the main DNA of a bacterium.
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  • DNA Libraries The total DNA in a cell is its genome A genomic DNA library is a collection of thousands of DNA fragments that represent all of the DNA in the genome A chromosome library contains all the DNA fragments in that specific chromosome A human genomic DNA library is stored in a collection of recombinant bacteria, each with a different fragment of DNA
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  • Producing a Genomic DNA Library or Chromosome Library
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  • Fig. 15-4, p. 327 Sites of cleavage Fragment 1 Fragment 2 Fragmen t 3 Fragment 4 Human DNA Produce recombinant DNA Gene for resistance to antibiotic Transformation RRR R Bacterium without plasmid Bacteria without plasmid fail to grow. Bacteria with plasmid live and multiply to form a colony. Plate with antibiotic- containing medium 1 2 3 4
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  • Locating a Sequence of Interest To identify a plasmid containing a sequence of interest, each plasmid is cloned until there are millions of copies A sample of bacterial culture is spread on agar plates so cells are widely separated each cell divides many times, forming a colony of genetically identical clones The next task is to determine which colony out of thousands contains the fragment of interest
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  • DNA Probes A segment of DNA that is homologous (identical) to part of the sequence of interest (DNA probe) can be used to detect the specific DNA sequence The DNA probe is a segment of single-stranded DNA that can hybridize (attach by base pairing) to complementary base sequences in target DNA DNA that is complementary to that particular probe is detected
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  • A cDNA Library It is possible to clone intact genes and avoid introns by using DNA copies of mature mRNA to construct complementary DNA (cDNA) Researchers use the enzyme reverse transcriptase to synthesize single-stranded cDNA, then DNA polymerase to make the cDNA double-stranded A cDNA library is formed using mRNA from a single cell type as the starting material
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  • Formation of cDNA
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  • Fig. 15-6, p. 329 ExonIntronExonIntronExon DNA in a eukaryotic chromosome Transcription Pre-mRNA RNA processing (remove introns) Mature mRNA Reverse transcriptase mRNA Mature mRNA cDNA copy of mRNA Degraded RNA cDNA DNA polymerase Double-stranded cDNA 1 2 3 4 5
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  • The Polymerase Chain Reaction The polymerase chain reaction (PCR) can be used to amplify a tiny sample of DNA without cloning in a cell PCR uses a heat-resistant DNA polymerase (Taq polymerase), nucleotides and primers to replicate a DNA sequence in vitro Cycles of denaturing (heating) and replication double the number of cloned molecules with each cycle
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  • The Polymerase Chain Reaction
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  • Using PCR PCR enables researchers to amplify and analyze tiny DNA samples from a variety of sources, ranging from crime scenes to archaeological remains Example: Investigators have used PCR to analyze mitochondrial DNA obtained from the bones of Neandertals
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  • KEY CONCEPTS 15.1 Scientists use DNA technology to produce many copies of specific genes (gene cloning)
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  • 15.2 DNA ANALYSIS LEARNING OBJECTIVES: Distinguish among DNA, RNA, and protein blotting
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  • Gel Electrophoresis Gel electrophoresis is used to separate mixtures of certain macromolecules: proteins, polypeptides, or DNA fragments Nucleic acids migrate through the gel toward the positive pole of the electric field because they are negatively charged due to their phosphate groups DNA fragments are separated by size small molecules move farther than large molecules
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  • Gel Electrophoresis
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  • Fig. 15-8a, p. 332 Standards of known sizes placed in well Direction of movement Mixtures placed in well Gel Buffer solution + 1
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  • Fig. 15-8b, p. 332 Anode Longer molecules Shorter molecules Cathode 2
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  • Fig. 15-8c, p. 332 3
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  • Southern Blot DNA separated by gel electrophoresis is denatured and transferred to a membrane, which picks up DNA like a blotter picks up ink this Southern blot is a replica of the gel The blot is incubated with a DNA probe, which hybridizes with any complementary DNA fragments the probe is detected by autoradiography