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Lecture 18

BISC403 Lecture 18: Lecture 18

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University of Delaware
Biological Sciences
Olabisi Oyenike

Lecture XVIII Chapter 9: Digital Analysis of Genomes I. Tools of molecular biology a. Human genome project an accurate sequence of the human genome was completed in 2003 b. By 2013, the genomes of >7000 species have been sequenced c. The general ideas behind genome sequencing are simple; i. Fragmenting the genome ii. Cloning DNA fragments iii. Sequencing DNA fragments iv. Reconstructing the genome sequence from fragments v. Analyzing the information found in genome d. Fragment because genome is huge e. How do we clone them into bacterial plasmids? f. Sequencing the genome have to know the code II. 9.1 Fragmenting DNA a. Classic way Restriction enzymes b. Rough way shearing the pieces c. Calculate the size of fragments d. Separating the fragments III. Serendipity, discovery of restriction endonucleases a. Experimenting with bacterial cells; pathogens that infect the bacteria b. Lawn of bacteria on a dish; infect with the viruses c. Bursts open from viruses that infected it d. Viral plaques e. Viruses grew abundantly on one strain of bacteria (lots of viral plaques) but not the other IV. Restriction endonucleases are enzymes that originate from bacteria protecting themselves against phages a. Few plaques had resistance mechanisms in place; restricted the phage from infecting the bacteria b. Fragmented the DNA into pieces c. In bacterial cells, methylation protected DNA from being cut; the virus cannot do that to its DNA, so the virus is cut up d. Restricts replication of viral genome V. Restriction enzymes fragment the genome at specific sites a. Dont just cut randomly; recognize specific sequences; these are few in bacteria b. Each restriction enzyme recognizes a specific sequence of bases anywhere within the genome i. Cuts sugarphosphate backbones of both strands ii. Restriction fragments are generated by digestion of DNA with restriction enzymes iii. Sequence has to be palindromic from the opposite strand; mirror image sequence c. Hundreds of restriction enzymes now available
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