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Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences Cutting and Joining DNA Fragments Restriction Enzyme: cleaves DNA at specific sequences DNA Ligase: joins DNA fragments by sealing nicks in backbone Use of the Restriction Enzyme Bam H1 5’— G G A T C C — 3’ 3’— C C T A G G — 5’ Results in 5’— G 3’— C C T A G sticky end G A T C C — 3’ G — 5’ sticky end DNA fragments with complementary sticky ends can associate to form new DNA combinations. Viewing DNA Fragments: Agarose Gel Electrophoresis Locating DNA Fragments by Southern Blotting Probe is complementary to DNA of interest DNA Fragments are transferred to membrane A subset of fragments binds to the probe Cloning Genes • Gene cloning = isolation of a specific segment of DNA in a form that can be duplicated in another cell • Requires a – Vector = carrier of DNA segment – Host = cell in which foreign DNA can be duplicated and its gene product may be synthesized • Recombinant DNA = DNA produced by joining fragments from two or more different sources Cloning Vectors • For Bacterial Hosts – Plasmid ampR Bam H1 tetR ampR tetR gene disrupted, no longer functional Cloning Vectors • For Bacterial Hosts – Bacteriophage – Cosmid – Expression Vector Cloning Vectors • For Eukaryotic Hosts – Shuttle Vector – YAC = yeast artificial chromosome – Ti plasmid Tools for Finding Genes • DNA library = collection of clones from one DNA donor – Categorized by: vector, source of DNA – Genomic library = all of the sequences from the genome of a single organism – cDNA library= complementary DNA, made using mRNA as a template Producing a Genomic Library Source of DNA for Library Many recombinant molecules are formed, each with a different DNA insert Producing cDNA Primer binds to poly-A tail DNA produced from RNA template Partial digestion of RNA template Second DNA strand produced using remaining RNA segments as primers Gaps in backbone sealed Applying Your Knowledge 1. 2. 3. 4. Genomic Library cDNA Library Either a genomic or cDNA library Neither a genomic nor cDNA library Which type of library would you use • To study transcribed gene sequences? • To study regulatory regions that are not part of genes? Tools for Finding Genes Probe: sequence of DNA or RNA that is complementary to the gene of interest; Used to locate a copy of the gene in a library by hybridization AATGCCAT TTACGGTA Denature DNA by heating Add Probe Probe Binds to gene AATGCCAT TTACGGTA TTACGGTA Screening a Library with a Probe Applying Your Knowledge 1. 2. 3. 4. 5. Probe Clone Vector Restriction Enzyme DNA Ligase A. An enzyme that cleaves DNA at specific sequences is a __________ . B. A sequence of DNA that is complementary to the gene of interest is a _________. C. A carrier molecule used to transport foreign DNA into a host cell is a __________. Amplifying DNA with the Polymerase Chain Reaction (PCR) • Copies a specific region in the DNA • Can be used with small amounts of sample • Uses repeated cycles of heating to denature DNA and cooling to synthesize new DNA • Requires – primers to begin synthesis – heat-stable polymerase (Taq polymerase) Polymerase Chain Reaction DNA Sequencing by the Sanger Method Relies on Chain Termination during replication due to incorporation of Dideoxynucleotides OH H H H deoxyribose dideoxyribose Replicate unknown DNA using dideoxynucleotides to terminate growing chains at various positions Unknown Sequence 3’-A-G-C-C-C-A-G-A-T-T-5’ Complementary Products Black= dideoxynucleotide 5’-T-3’ 5’-T-C-G-G-G-3’ 5’-T-C-G-3’ 5’-T-C-G-G-G-T-C-T-3’ 5’-T-C-3’ 5’-T-C-G-G-G-T-C-T-A-3’ 5’-T-C-G-G-G-T-C-T-A-A-3’ 5’-T-C-G-G-G-T-C-3’ 5’-T-C-G-G-G-T-3’ 5’-T-C-G-G-3’ Use gel electrophoresis to separate replicated fragments that differ by one base A Dideoxynucleotide T C G Size of DNA on gel shows distance from start of replication Labeling of dideoxynucleotide shows complementary sequence Determine unknown sequence from complementary sequence with base-pairing rules A Dideoxynucleotide T C G Complementary Sequence 5’-T-C-G-G-G-T-C-T-A-A 5’-T-C-G-G-G-T-C-T-A 5’-T-C-G-G-G-T-C-T 5’-T-C-G-G-G-T-C 5’-T-C-G-G-G-T 5’-T-C-G-G-G 5’-T-C-G-G 5’-T-C-G 5’-T-C 5’-T Complementary Sequence Unknown Sequence 5’-T-C-G-G-G-T-C-T-A-A-3’ 3’-A-G-C-C-C-A-G-A-T-T-5’ Applications of Recombinant DNA Technology Genetic Testing and Gene Therapies Products of Biotechnology RFLP Analysis • RFLP = Restriction Fragment Length Polymorphism • Identifies differences in the length of restriction fragments derived from similar DNA sequences • Analyzed by Southern Blotting • Used in gene mapping RFLP Analysis in Paternity Testing X X X X X X X X RFLP Analysis in Forensics 1 2 3 Suspects CS RC IE MN EE 4 5 67 Suspects DNA Fingerprinting • Detects patterns for short repeated sequences that are highly variable among humans • Described as – VNTR: Variable Number of Tandem Repeats – STR: Short Tandem Repeats – Microsatellites Analysis using Microsatellite sequences Genetic Testing On the basis of this analysis, the genotype of the fetus is 1. AS 2. AA 3. SS 4. Unknown Gene Therapy Andrew Gobea was born with SCID, an immune deficiency due to a defect in the ADA gene. Four days after his birth, he received an injection of his own stem cells carrying functional ADA genes. Andrew’s immune system began producing ADA, but the amount was not sufficient to maintain full health. He needs daily injections of ADA enzyme. RNA Interference as a Possible Treatment for High Cholesterol • High levels of ApoB, a protein component of blood lipid carriers, are associated with high cholesterol levels • In monkeys, siRNA for ApoB has lowered the mRNA for ApoB in liver cells and also reduced serum cholesterol levels siRNA for ApoB was delivered in lipid capsules Recombinant DNA Products Pharmaceutical Used for Factor VIII Blood Clotting Human Growth Hormone Pituitary Dwarfism Insulin Diabetes Interferon Cancer Tissue Plasminogen Activator Vaccine Heart Attack Hepatitis B Genetically Modified Organisms Introducing or modifying specific genes to alter the phenotype of an organism Transgenic: organism that contains a gene from another species in all of its cells Transgenic Animals Models of Human Disease Produce Pharmaceuticals Studying Gene Function with Knockout Mice • neo+ gene gives resistance to G418 • tk+ gene gives sensitivity to gancyclovir • Insertion of neo+ into target gene yields neo+ and tkcells, resistant to G418 and to gancyclovir Transgenic Plants “Golden” rice with beta-carotene and extra iron Round Up Ready Soybeans are resistant to herbicide Bt Corn produces its own pesticide What Are Some Concerns?