LS16Restrictionenzyme

LS16Restrictionenzyme - Lecture 16: DNA sequencing &...

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1 Lecture 16: DNA sequencing & Restriction Enzymes DNA sequencing Restriction Enzymes Restriction Mapping Recombinant DNA technology Studies in molecular biology rely on a number of experimental technologies that are collectively called recombinant DNA technology. Some important ones are: 1. DNA sequence 2. Cutting and joining of DNA fragments 3. PCR 4. Gene cloning 5. DNA and RNA hybridization analyses 6. Recombinant protein expression techniques 7. Transgenic techniques (organism cloning)
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2 Landmark Breakthroughs in DNA Technology •Bacterial transformation methods - Early 1970’s •Gene transfection into mammalian cells, Graham & Van der Erb - 1973 •First functional recombinant gene expressed in E. coli •Discovery of restriction enzymes & use in DNA mapping, Hamilton Smith, Werner Arber, Dan Nathans - 1978 Nobel Prize •Recombinant DNA, Paul Berg - 1980 Nobel Prize •DNA sequencing, Walter Gilbert, Frederick Sanger - 1980 Nobel Prize •Polymerase Chain Reaction (PCR), Kerry Mullis - 1993 Nobel Prize Recall some properties of Nucleic Acids 1. DNA and RNA are soluble in aqueous (water) solutions, but insoluble in 75% (and up) ethanol. DNA or RNA be can be extracted from tissue using aqueous solutions, and precipitated by addition of ethanol to the aqueous solution . 2. Double-strand DNA can be denatured, the denatured DNA can be renatured. Formation of a dsDNA from two ssDNAs coming from two different parental dsDNAs is called hybridization. Hybridization can also occur between a ssDNA and a RNA. 3. DNA and RNA have phosphate group at 5’ ends, and OH at 3’ ends. DNA and RNA are usually negatively charged. DNA and RNA migrate according to their size in the agarose gel during electrophoresis (smaller ones migrate faster, larger ones run slower). 4. A fluorescent dye ethidium bromide (EtBr) can bind DNA (and RNA) by inserting (intercalating) between adjacent bases in single-strand or double-strand DNA and RNA molecules. EtBr can cause the double helix to unwind and it is a carcinogen (agents causing cancer).
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3 1. DNA sequence analysis The first DNA sequencing method used is a chemical method known as Maxam-Gilbert method. The most widely used method of DNA sequence analysis is the “ Sanger method or dideoxy chain-termination method ”, which relies on the in vitro DNA replication reaction catalyzed by a DNA polymerase. Gilbert and Sanger shared the Nobel price in
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This note was uploaded on 01/16/2010 for the course LIFESCI Life scien taught by Professor Lorenz,t.c. during the Fall '09 term at UCLA.

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LS16Restrictionenzyme - Lecture 16: DNA sequencing &...

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