19.1 techniques of molecular genetics have revolutionized biology
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19.1 Techniques of Molecular Genetics Have Revolutionized Biology. Recombinant DNA Technology — Genetic Engineering — Biotechnology: Locating, isolating, altering, and studying DNA segments Biotechnology: Using recombinant DNA technology to develop new biological products. - PowerPoint PPT PresentationTRANSCRIPT
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19.1 Techniques of Molecular Genetics Have Revolutionized Biology
• Recombinant DNA Technology—Genetic Engineering—Biotechnology:
• Locating, isolating, altering, and studying DNA segments
• Biotechnology:
• Using recombinant DNA technology to develop new biological products
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19.2 Molecular Techniques Are Used to Isolate, Recombine, and Amplify Genes
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Cutting and Joining DNA Fragments
• Restriction enzymes: recognizing and cutting DNA at specific nucleotide sequences– Palindromic sequences– Immune system of bacteria
• Type II restriction enzyme: most useful enzyme
• By adding methyl groups to the recognition sequence to protect itself from being digested by its own enzyme in bacteria
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Cutting and Joining DNA Fragments
• Cohesive ends: fragments with short, single-stranded overhanging ends
• Blunt ends: even-length ends from both single strands
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Viewing DNA Fragments
• Gel electrophoresis-separation of DNA fragments by size through a gel medium• Smaller fragments migrate faster
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Viewing DNA Fragments
• Locating DNA fragments with Southern blotting and probes
• Probe: DNA or RNA with a base sequence complementary to a sequence in the gene of interest• Is usually labeled for easy detection
• Radioactive P32
• Fluorescent tag
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Cloning Genes
• Gene cloning: amplifying a specific piece of DNA via a bacteria cell
• Cloning vector: a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell– Has features that make it easier to insert DNA and
select for presence of vector in cell.• Origin of replication• Antibiotic resistance gene• Cloning site
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Cloning Genes
• Plasmid vectors
• Linkers: synthetic DNA fragments containing restriction sites
• Transformation of host cells with plasmids
• Selectable markers are used to confirm whether the cells have been transformed or not.
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Amplifying DNA fragments with the polymerase chain reaction (PCR)
• Taq polymerase: stable DNA polymerase at high temperature
• Researcher designs specific oligonucleotide primers that serve as the ends of the amplified fragment
• Very similar to replication
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19.3 Molecular Techniques Can Be Used to Find Genes of Interest
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Gene Libraries
• Gene library: a collection of clones containing all the DNA fragments from one source
• Creating a genomic DNA library
• cDNA library: consisting only of those DNA sequences that are transcribed into mRNA
• Creating a cDNA library
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Gene Libraries
• Screening DNA libraries
• Plating clones of the library
• Probing plated colonies or plaques
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In Situ Hybridization
• DNA probes used to determine the chromosomal location and to visualize a gene while it is in a cell
• May also be used to detect localization of mRNA expression in multicellular organisms.
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• Positional Cloning• Isolating genes on the basis of their position
on a genetic map
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19.4 DNA Sequences Can Be Determined and Analyzed
• Restriction Fragment Length Polymorphisms (RFLPs)
• Some DNA fragments have different restriction sites due to mutation for the same restriction enzyme, which causes polymorphisms within a population.
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DNA Sequencing
• Sanger’s dideoxy-sequencing method
• Dideoxyribonucleoside triphosphate (ddNTP) lacks a 3′-oh group, which terminates DNA synthesis.
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DNA Fingerprinting (DNA Profiling)
• Microsatellites: variable number of copies of repeat sequences possessed by many organisms, which can be amplified by PCR
Combined with RFLP analysis to form more thorough fingerprint
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19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function
• Forward genetics: – Begins with a phenotype to a gene that
encodes the phenotype
• Reverse genetics:– Begins with a gene of unknown function, first
inducing mutations and then checking the effect of the mutation on the phenotype
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19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function
• Site-directed mutagenesis
• Reverse genetics: creating mutation in particular DNA sequences, and then studying the effects of these mutation on the organisms
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19.5 Molecular Techniques Are Increasingly Used to Analyze Gene Function
• Silencing genes with RNAi
• Using RNAi for the treatment of human disease: lowering ApoB with RNAi
• Model Genetic Organism
• The mouse, Mus musculus
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19.6 Biotechnology Harnesses the Power of Molecular Genetics
• Pharmaceuticals• Human insulin• Specialized bacteria• Agricultural products• Oligo nucleotide drugs• Genetic testing• Gene therapy• Direct transfer of genes into humans to
treat disease