L12-13 - 1 Biochemistry Sixth Edition Chapter 5 Exploring Genes and Genomes Copyright © 2007 by W H Freeman and Company Berg • Tymoczko •

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Unformatted text preview: 1 Biochemistry Sixth Edition Chapter 5: Exploring Genes and Genomes Copyright © 2007 by W. H. Freeman and Company Berg • Tymoczko • Stryer Biological processes involve changes in gene expression patterns e.g. metamorphosis •Gene expression can be followed on microarrays. •Pattern changes with changes in biological processes ( e.g. transcription) 5.1) The exploration of genes relies on key tools 1) Restriction enzyme analysis • See New England Biolabs <http://www.neb.com> 2) Blotting techniques 3) DNA sequencing • At Penn State, <http://www.huck.psu.edu/stf/naf/Sequencing.html> 4) Solid-phase synthesis of nucleic acids • At Penn State, <http://www.huck.psu.edu/stf/naf/Synthesis.html> 5) The polymerase chain reaction • Interview with Kary Mullis, inventor of PCR and 1993 Nobel Prize in Chemistry: <http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis- interview.html> 2 1.) Restriction enzymes are DNA scissors Sac II (from S treptomyces ac hromogenes ) in this example 5’CCGC pGG3’ 3’GGp CGCC5’ 2-fold A wide variety of restriction enzymes are commercially available Overhang: 5’ (sticky ends) 5’ (sticky ends) none (Fush ends) 3’ (sticky ends) 5’ (sticky ends) •‘Sticky ends’ are aka ‘cohesive ends’ •‘¡lush ends’ are aka ‘blunt ends’ Restriction digests of a given DNA can be separated by gels Restriction enzyme: Each enzyme (A, B, C) gives a unique ¢ngerprint. •¡or shorter DNA (<1 kb), use polyacrylamide gels (PAGE). Denaturing. •¡or longer DNA (up to 20 kb), use agarose gels. non- denaturing •¡or very long DNA (1 Mb+), use P¡GE (p ulse f ield g el e lectrophoresis) 3 2.) Blotting techniques: Southern blotting ≈ 20mer radiolabeled ‘*_____’ •Southern blotting was developed by Edwin Southern •Northern blotting is a way to detect an RNA fragment with 32 P-DNA •Western blotting detects proteins with an antibody (see earlier chapters) 3.) Sanger sequencing of DNA •aka ‘dideoxy’ or ‘chain termination’ method •Nobel prize in Chemistry (1980) •Need to run a total of 4 reactions: ddATP, ddCTP, ddGTP, ddTTP molecule 1 molecule 2 (molecule 1 ≠ molecule 2) (ddATP is doped in) •missing ‘HO’ •chain has been ‘terminated’ :OH (3’OH of preceding base) 4 DNA sequencing is automated •DNA sequencing is now done in one pot with fuorescently labeled ddNTP ( ddATP , ddCTP , ddGTP, ddTTP ) •Need to do only 1 (not 4) labeling reactions •Get sequence From 1 (not 4) lanes in a gel •Can sequence 800+ bp at a time (much more eF¡cient than protein sequencing!) 4.) Solid-phase synthesis oF DNA (RNA) All nucleophiles are blocked. Each base is protected at two positions: 1.) The 5’-OH 2.) The base itselF 3.) ¢or RNA synthesis, must protect 2’OH too!...
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This note was uploaded on 07/23/2008 for the course CHEM 476 taught by Professor Bevilacqua,philip during the Fall '07 term at Pennsylvania State University, University Park.

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L12-13 - 1 Biochemistry Sixth Edition Chapter 5 Exploring Genes and Genomes Copyright © 2007 by W H Freeman and Company Berg • Tymoczko •

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