CAMPBELL BIOLOGY TENTH EDITION Reece Urry Cain Wasserman Minorsky Jackson 20 DNA Tools and Biotechnology Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick 2014 Pearson Education Inc Biotechnology is the manipulation of organisms or their components to make useful products The applications of DNA technology affect everything from agriculture to criminal law to medical research 2014 Pearson Education Inc Concept 20 1 DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry The complementarity of the two DNA strands is the basis for nucleic acid hybridization the base pairing of one strand of nucleic acid to the complementary sequence on another strand Genetic engineering is the direct manipulation of genes for practical purposes 2014 Pearson Education Inc DNA Sequencing Researchers can exploit the principle of complementary base pairing to determine a gene s complete nucleotide sequence called DNA sequencing The first automated procedure was based on a technique called dideoxy or chain termination sequencing developed by Sanger 2014 Pearson Education Inc Figure 20 2 a Standard sequencing machine b Next generation sequencing machines 2014 Pearson Education Inc Figure 20 3 Technique DNA template strand 5 C T G A C T T C G A C A 3 A 5 Primer 3 T G T T 5 DNA polymerase dATP ddATP dCTP ddCTP dTTP ddTTP dGTP ddGTP P P P Shortest dd T G A A G C T G T T Longest labeled strand Detector Laser Shortest labeled strand Results Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand 2014 Pearson Education Inc P P P G Labeled strands DNA template C strand T G A dd G C dd A A T dd A T A A C G G G 3 dd G G dd C C C C C A T T T T T C G G G G G A T T T T T 3 A T T T T 5 T Direction of movement of strands Dideoxyribonucleotides fluorescently tagged Deoxyribonucleotides G A C T G A A G C dd C T G A A G C T G T T dd A C T G A A G C T G T T G dd G 3 A C T G A A G C T G T 5 T Longest Next generation sequencing techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments Thousands or hundreds of thousands of fragments 400 1 000 nucleotides long are sequenced in parallel This is a type of high throughput technology 2014 Pearson Education Inc Figure 20 4 Technique 1 Genomic DNA is fragmented Results 4 mer 2 Each fragment is isolated with a bead 3 mer A T G C 2 mer 3 Using PCR 106 copies of each fragment are made each attached to the bead by 5 end 1 mer 4 The bead is placed into a well with DNA polymerases and primers Template strand of DNA 5 3 5 3 Primer A T GC 5 A TGC DNA polymerase Template C strand C of DNA A A dATP T G TA PPi GC GC AG Primer TA 6 If a nucleotide is joined to a growing strand PPi is released causing a flash of light that is recorded 2014 Pearson Education Inc A solution of each of the four nucleotides is added to all wells and then washed off The entire process is then repeated A T GC C C A dTTP A T G TA GC GC AG TA 7 If a nucleotide is not complementary to the next template base no PPi is released and no flash of light is recorded A TGC C C A dGTP A T G TA GC GC AG TA A T GC C C A A T GC TA GC GC AG TA dCTP PPi 8 The process is repeated until every fragment has a complete complementary strand The pattern of flashes reveals the sequence Making Multiple Copies of a Gene or Other DNA Segment To work directly with specific genes scientists prepare well defined DNA segments in multiple identical copies by a process called DNA cloning Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome Researchers can insert DNA into plasmids to produce recombinant DNA a molecule with DNA from two different sources 2014 Pearson Education Inc Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA This results in the production of multiple copies of a single gene The production of multiple copies of a single gene is a type of DNA cloning called gene cloning 2014 Pearson Education Inc Figure 20 5 Bacterium Cell containing gene of interest 1 Gene inserted Bacterial chromosome into plasmid Plasmid Gene of interest Recombinant DNA plasmid DNA of chromosome foreign DNA 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the cloned gene of interest Gene of interest Protein expressed from gene of interest Copies of gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste 2014 Pearson Education Inc Protein harvested 4 Basic research and various applications Human growth hormone treats stunted growth Protein dissolves blood clots in heart attack therapy A plasmid used to clone a foreign gene is called a cloning vector Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes 2014 Pearson Education Inc Using Restriction Enzymes to Make a Recombinant DNA Plasmid Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites A restriction enzyme usually makes many cuts yielding restriction fragments The most useful restriction enzymes cut DNA in a staggered way producing fragments with sticky ends 2014 Pearson Education Inc Sticky ends can bond with complementary sticky ends of other fragments DNA ligase is an enzyme that seals the bonds between restriction fragments 2014 Pearson Education Inc Figure 20 6 Bacterial plasmid Restriction site 5 3 G AAT T C C T TAAG DNA 3 5 1 Restriction enzyme cuts the sugar phosphate backbones at each arrow 3 G C T TA A 5 5 3 Sticky end 2 Base pairing of sticky ends produces various combinations 5 3 3 DNA ligase 5 A AT T C G 3 3 5 G AAT T C C T TA A G 5 3 5 A AT T C G 3 3 5 3 G C T TA A 5 Fragment from different DNA molecule cut by the same restriction enzyme 3 5 G AAT T C C T TA A G 5 3 3 5 One possible combination seals the strands 5 3 3 Recombinant DNA molecule Recombinant plasmid 2014 Pearson Education Inc 5 To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme To separate and visualize the fragments produced gel …
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