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Download Chapter 8: Recombinant DNA Technology 1. Tools of Recombinant
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Chapter 8: Recombinant DNA Technology 1. Tools of Recombinant DNA Technology 2. PCR & DNA Sequencing 1. Tools of Recombinant DNA Technology Restriction enzyme recognition sequence Restriction Enzymes GAATTC CTTAAG DNA 1 Restriction enzyme cuts the DNA into fragments 2 G CTTAA The cutting and splicing of DNA in vitro involves the use of restriction enzymes (RE’s): A AT TC G Sticky end Addition of a DNA fragment from another source A AT TC G 3 Two (or more) fragments stick together by base-pairing 4 G A AT T C C T TA A G G A AT T C C T TA A G DNA ligase Pastes the strand 5 Recombinant DNA molecule G CTTA A e.g. EcoR I cuts at: ..GAATTC.. ..CTTAAG.. Hind III cuts at: ..AAGCTT.. ..TTCGAA.. There are many different RE’s, each cutting a different sequence 1 “Cloning” a Gene DNA of interest is inserted into a plasmid & carried inside bacterial “clone”: 1) cut plasmid and DNA to be cloned with same RE 2) ligate fragments together using DNA ligase enzyme 3) transform bacteria, select for “clones” with plasmid **Allows DNA of interest to be easily “grown” and purified** Bacterial cell Bacterial chromosome DNA containing gene of interest Plasmid 1 Isolate plasmid. Gene of interest 4 2 Enzymatically cleave DNA into fragments. 3 Isolate fragment with the gene of interest. Insert gene into plasmid. 5 Insert plasmid and gene into bacterium. 6 Culture bacteria. Harvest copies of gene to insert into plants or animals Harvest proteins coded by gene Create Eliminate beneficial undesirable phenotypic combination of traits traits Produce vaccines, antibiotics, hormones, or enzymes 2 Cloning Vectors DNA designed to carry “DNA of interest” within another organism (or virus): • plasmids (bacteria) , viral DNA, artificial chromosomes (yeast) Plasmid cloning vectors contain: • convenient restriction sites • origin of replic. • antibiotic resistance genes for selection 2. PCR & DNA Sequencing PCR & DNA sequencing are techniques that involve manipulating DNA replication in vitro… 3 Each technique requires the following: 1) Source of DNA of Interest • tissue sample that may contain pathogenic DNA (PCR) • “cloned” DNA from species of interest inserted into a vector (plasmid, virus, artificial chromosome) 2) Artificial (man-made) Primer(s) • ~20 nt DNA fragment that targets DNA synthesis to region of interest 3) Raw Materials for DNA Synthesis • nucleotides (dNTP’s), DNA polymerase a. PCR Overview of PCR Every PCR (Polymerase Chain Reaction) reaction requires the following: 1) source of target DNA template 2) artificial primers “flanking” DNA of interest 3) heat-stable DNA polymerase (from hyperthermophile) 4) dNTP’s 5) automated thermocycler to facilitate repeated: • denaturation of DNA (separating the 2 strands) • hybridization of primers to template • DNA synthesis 4 Specificity of PCR • primers must be specific for DNA of interest • complementary to target DNA • if target DNA is present, the sequence flanked by the primers will be selectively amplified Illustration of PCR First cycle Gene of interest 3′ 5′ 5′ 3′ Heat to 94°C 3′ Third cycle Fourth cycle 2 double strands 5′ Denaturation 1 5′ Second cycle Original DNA molecule with gene of interest 4 double strands 3′ dNTPs 2 Priming DNA primer 8 double strands DNA polymerase (heat stable) 3′ 5′ 5′ 3′ DNA polymerase heat-stable DNA polymerase must remain active after heating to 94o C Cool to 65°C 16 double strands 5′ 3′ DNA primer 3′ 5′ 3 Extension at 72°C Isolation of PCR DNA Fragments The resulting PCR products can be separated by size via gel electrophoresis: (–) Wells Electrophoresis chamber filled with buffer solution E A B D C a DNA (50) (40) (35) Agarose gel (+) (15) (10) (5) Movement of DNA b Wire Lane of DNA fragments of known sizes (kilobase pairs) • once purified from the gel, PCR fragments can be cloned into vectors (plasmids) and sequenced to verify! 5 b. DNA Sequencing DNA Sequencing uses Chain Terminators Normal nucleotide (dNTP) Dideoxynucleotide (ddNTP) * 4 separate DNA synthesis reactions are carried out, each containing: • DNA template to be sequenced • dNTP’s • DNA primer (fluorescently labeled) • DNA polymerase Each of the 4 reactions is spiked with a different labeled ddNTP: • ddATP, ddCTP, ddGTP or ddTTP • primers used for each reaction labeled with a different color fluorescent marker 6 “A” reaction • termination of DNA synthesis from the primer will occur at random “A’s” due to ddATP “C” reaction • termination of DNA synthesis from the primer will occur at random “C’s” due to ddCTP “G” reaction • termination of DNA synthesis from the primer will occur at random “G’s” due to ddGTP “T” reaction • termination of DNA synthesis from the primer will occur at random “T’s” due to ddTTP + “blue” primer + “green” primer + “red” + “yellow” primer primer Resolving the DNA Fragments Separation of the resulting labeled DNA fragments from each reaction by gel electrophoresis reveals the sequence! • each labeled DNA strand begins at the primer • the length of each fragment depends on where the strand terminated • i.e., where the ddNTP was added, thus causing chain termination • shorter DNA fragments move faster through the gel, longer fragments move more slowly **The relative lengths of the fragments from each reaction reveals the overall DNA sequence** 7 radioactively labeled fluorescently labeled Key Terms for Chapter 8 • restriction enzyme • “cloning” of a gene, cloning vector • gel electrophoresis • dideoxynucleotide triphosphates (ddNTPs) • chain terminators Relevant Chapter Questions MC: 5, 8 T/F: 1-3 8