Chapter 5 Biochemistry6e

Chapter 5 Biochemistry6e - Chapter 5 Biochemistry Exploring...

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Chapter 5 Biochemistry Exploring Genes and Genomes - 6 th Edition by Berg, Tymoczko and Stryer Progress in biotechnology has been facilitated by the discovery and development of a number of molecular biology tools and techniques. Among the most useful are: Restriction Enzyme Analysis - Restriction enzymes are endonucleases that precisely cut the DNA so that fragments of DNA are made. Blotting Techniques - Southern blots separate and characterize DNA fragments, Northern blots do the same for RNA while Western blots are used to separate and characterize proteins using antibodies. DNA Sequencing - Rapid, efficient methods of determining the precise sequence of DNA have been developed. Solid-Phase Sequencing of Nucleic Acids - Exact sequences of nucleic acids can be made from scratch and used to identify other nucleic acids. The Polymerase Chain Reaction (PCR) - The polymerase chain reaction allows a very minute amount of DNA, perhaps even a single copy , to be copied and synthesized millions to billions of times, thereby allowing ample amounts of the targeted DNA so that sequencing and other manipulations can be accomplished. The one tool that allows all of these techniques to be coordinated is the computer. The computer allows the means by which vast amounts of data can be stored and manipulated. Restriction Endonucleases (Restriction enzymes) - These enzymes recognize and cleaves specific sequences in a double-stranded DNA helix. It is the phosphodiester bond of the sugar-phosphate backbone that is hydrolyzed. Restriction endonucleases are made by a large number of prokaryotes that avoid destroying their own DNA because they methylate those sites that are cleaved by the restriction endonuclease, an alteration that makes the restriction endonuclease unable to cut the DNA. Most of the sites of the restriction endonucleases contain two-fold rotational symmetry . This 1
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means that the recognized site is a palindrome (inverted repeat) with the cleavage site being symmetrically partitioned (Restriction Cutting Figure and Figure 5.1) . Note that it the 3' side of the symmetry axis where the cleavage occurs. Restriction endonucleases allow pieces of DNA to be generated that are more manageable than what is otherwise available. For instance, the genome of a virus of 7.5 kb can be cut into smaller fragments by the use of restriction endonucleases, either individually or when two or more are used collectively. Gel electrophoresis is used to separate pieces of DNA, such as those generated by restriction endonuclease treatment. The mobility of a piece of DNA is inversely proportional, up to a limit, to the logarithm of the number of bases in the DNA. Fragments of <1000 bp.s are separated by polyacrylamide gels while agarose gels, which are more porous, can separate from about 1-20 kb (Show Figure 5.2) .
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