Recombinant DNA Chapter 18. Learning Objectives Define Clone and DNA Cloning List the three steps of production of recombinant DNA Describe the characteristics

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Text of Recombinant DNA Chapter 18. Learning Objectives Define Clone and DNA Cloning List the three steps of...

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  • Recombinant DNA Chapter 18
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  • Learning Objectives Define Clone and DNA Cloning List the three steps of production of recombinant DNA Describe the characteristics and uses of a restriction endonuclease Diagram the process of identifying a transformed bacterial colony containing a gene of interest using Ampicillin Resistance, Lactose Metabolism plasmids and nucleic acid hybridization probes
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  • Learning Objectives Explain the uses of RFLPs Describe the process of producing a transgenic organism, and explain its usefulness
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  • DNA cloning Clone: genetically identical cells or individuals derived from a single ancestor DNA cloning: a method of producing a large amount of the DNA of interest Large amounts of identical pieces of DNA enable us to manipulate and recombine genetic material
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  • DNA Technologies DNA technologies are used in molecular testing for many human genetic diseases DNA fingerprinting used to identify human individuals and individuals of other species Genetic engineering uses DNA technologies to alter the genes of a cell or organism DNA technologies and genetic engineering are a subject of public concern
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  • Recombinant DNA DNA from two or more sources joined together DNA of interest can be spliced into bacterial plasmids (recombination) Plasmids replicate (amplification) Plasmids (DNA) are extracted (isolation)
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  • Endonucleases Restriction enzymes (endunucleases) cut DNA at specific sequences in restriction sites Restriction fragments result Sticky ends have unpaired bases at cuts which will hydrogen bond Ligase stitches together paired sticky ends
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  • Fig. 18-3, p. 374 Restriction site for EcoRI DNA Sticky end Another DNA fragment produced by EcoRI digestion Sticky end Nick in sugarphosphate backbone Recombinant DNA molecule EcoRI restriction enzyme cleaves sugarphosphate backbones at arrows. DNA fragments with the same sticky ends can pair. Shown here is a DNA fragment inserting between two other DNA fragments, as happens when inserting a DNA fragment into a bacterial plasmid. Nicks in sugar phosphate backbones are sealed by DNA ligase. 1 2 3
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  • Recombinant DNA Restriction endonucleases Each type is specific for a four to eight base pair long palindromic recognition sequence of DNA Palindrome- reads the same on each strand 3 to 5 like GAATTC CTTAAG
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  • Fig. 18-4a, p. 375 Gene of interest DNA fragments with sticky ends Cell Restriction site Cut plasmid cloning vectors with a restriction enzyme to produce sticky ends Plasmid cloning vector amp R gene lacZ + gene
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  • Fig. 18-2b, p. 373 5 Introduce recombinant molecules into bacterial cells; each bacterium receives a different plasmid. As the bacteria grow and divide, the recombinant plasmids replicate, thereby amplifying the piece of DNA inserted into the plasmid. Identify the bacterium containing the plasmid with the gene of interest inserted into it. Grow that bacterium in culture to produce large amounts of the plasmid for experiments with the gene of interest. Inserted genomic DNA fragment Bacterium Bacterial chromosome Progeny bacteria Recombinant DNA molecules 4
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  • Recombinant DNA Break cells and use restriction enzyme to isolate DNA of interest (prokaryotic or eukaryotic) Insert into plasmid (recombination) Transform into bacteria (replication) Not very efficient, so for the third step (isolation)- you need to have engineered a way to find the bacteria of interest
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  • Four possibilities 1. Desired outcome: plasmid, lac+ broken, gene of interest inserted 2. Bacteria transformed with plasmid, but wrong gene inserted 3. Bacteria transformed with plasmid only- no gene at all inserted 4. Bacteria not transformed
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  • Fig. 18-4b, p. 375 Resealed plasmid cloning vector with no inserted DNA fragment Inserted DNA fragments with gene of interest Recombinant plasmids Inserted DNA fragment without gene of interest Nonrecombinant plasmid
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  • Recombinant DNA Insert into special screening plasmid- which contains the same restriction enzyme site used above, located in a lacZ gene. For recombination screening the lacZ gene is broken successfully, it will be white. If not, it will be blue. The plasmid also contain ampicillin resistance If transformation worked, the bacteria will grow on plates containing ampicillin. Those who were not transformed will not grow.
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  • Fig. 18-4c, p. 375 Bacteria not transformed with a plasmid Bacteria transformed with plasmids Selection: Transformed bacteria grow on medium containing ampicillin because of amp R gene on plasmid. Screening: Blue colony contains bacteria with a non- recombinant plasmid; that is, the lacZ + gene is intact. Plate containing ampicillin and X-gal Untransformed bacterium cannot grow on medium containing ampicillin. White colony contains bacteria with a recombinant plasmid; that is, the vector with an inserted DNA fragment. Once the white colony with the gene of interest is identified, it can be grown in culture to produce large quantities of the plasmid.
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  • DNA Hybridization Uses nucleic acid probe to identify gene of interest in set of clones Probe has tag for detection Identified colony produces large quantities of cloned gene
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  • Fig. 18-5a, p. 377 Replica of bacterial colonies Culture medium containing ampicillin Filter paper Bacterial colony
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  • Fig. 18-5b, p. 377 Labeled probe (single stranded) Bag Filter Labeled single- stranded DNA probe for the gene of interest Hybridization has occurred between the labeled probe and the plasmids released from the bacteria in this colony. The hybridization is detected in subsequent steps. Plasmid DNA (single stranded)
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  • Fig. 18-5c, p. 377 Original master plate Developed photographic film Corresponds to one colony on master plate
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  • 4 Possibilities OutcomeAMPLACPROBE Right GeneyesNoYes Wrong GeneYesNo Plasmid onlyYes n/a No PlasmidNo n/a
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  • How else do we use Restriction Endonuclease?
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  • RFLPs Restriction fragment length polymorphisms DNA sequence length changes due to varying restriction sites from same region of genome Sickle cell anemia has RFLPs Southern blot analysis uses electrophoresis, blot transfer, and labeled probes to identify RFLPs Alternative is PCR and electrophoresis
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  • Fig. 18-8, p. 381 -Globin gene 175 bp Normal allele Sickle-cell mutant allele Region of probe used to screen for sickle-cell mutation 201 bp 376 bp MstII
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  • DNA Fingerprinting Distinguishes between individuals Uses PCR at multiple loci within genome Each locus heterozygous or homzygous for short tandem repeats (STR) PCR amplifies DNA from STR Number of gel electrophoresis bands shows amplified STR alleles 13 loci commonly used in human DNA fingerprinting
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  • Forensics and Ancestry Forensics compares DNA fingerprint from sample to suspect or victim Usually reported as probability DNA came from random individual Common alleles between children and parents used in paternity tests Same principle used to determine evolutionary relationships between species
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  • Fig. 18-10a, p. 383 3 different alleles DNA a. Alleles at an STR locus Left PCR primer STR locus 9 repeats Right PCR primer 11 repeats 15 repeats
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  • Fig. 18-10b, p. 383 b. DNA fingerprint analysis of the STR locus by PCR Cells of three individuals Extract genomic DNA and use specific primers to amplify the STR locus using the PCR. CB A C B A 15,911,911,11 Anyalyze PCR product by gel electrophoresis Positions corresponding to alleles of STR locus 15 11 9
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  • Genetic Engineering Transgenic organisms Modified to contain genes from external source Expression vector has promoter in plasmid for production of transgenic proteins in E. coli Example: Insulin Protocols to reduce risk of escape
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  • Animal Genetic Engineering Transgenic animals used in research, correcting genetic disorders, and protein production Germ-line cell transgenes can be passed to offspring (somatic can not) Embryonic germ-line cells cultured in quantity, made into sperm or eggs Stem cells
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  • Fig. 18-11a, p. 385 Pure population of transgenic cells Germ-line cells derived from mouse embryo Transgene Cell with transgene
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  • Fig. 18-11b, p. 385 Mice have transgenic cells in body regions including germ line Genetically engineered offspringall cells transgenic
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  • Gene Therapy Attempts to correct genetic disorders Germ-line gene therapy cant be used on humans Somatic gene therapy used in humans Mixed results in humans Successes for adenosine deaminase deficiency (bubble kid) and sickle-cell Deaths from immune response and leukemia- like conditions http://history.nih.gov/exhibits/genetics/sect4.htm
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  • Animal Genetic Engineering Pharm animals produce proteins for humans Usually produced in milk for harmless extraction Cloned mamma