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Karen Williams

CH.8 summary: -an ambitious and expensive plan to sequence the human genome, the human genome project (HGP)m commenced in 1990. As part of the HGP, the genomes of several well studied model organisms in genetics were also sequences. A final version of the human genome sequence was released in 2003 -genomics is the study of the complete DNA sequence of an organism. The process starts with the cloning of an organism`s DNA into one of many types of vectors. Next, the exact sequence of nucleotides within these clones is generated. These sequence data can then be used in many further types of analyses, such as identifying which regions encode genes -DNA cloning is the introduction of foreign DNA sequences into a particular type of vector, an artificially constructed DNA molecule that allows the foreign DNA to be replicated when placed into a host cell. Usually a bacterium or yeast. Cloning entire chromosomes typically is impossible, so the genomic DNA of an organism typically must be broken down into smaller fragments before it can be clones. One way to cut DNA is through the use of restriction enzymes -Different kinds of cloning vectors have been developed; plasmids are the most commonly used. Cloning vectors typically replicate within or more host organisms, have restriction sites into which foreign DNA can be inserted, and have one or more selectable markers to use in selecting cells that contain the vectors. Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) enable DNA fragments several hundred kilo base pairs long to be cloned in E. coli and yeast, respectively -restriction enzymes cut DNA at specific locations called restriction sites. Each restriction enzyme recognizes a unique sequence of nucleotides within the DNA, the restriction site, and cleaves both strands of DNA, often producing a small overhang called “sticky end”. Complementary sticky ends can reannal with each other, bring together to completely different pieces of DNA to form a recombinant DNA molecule as long as they have both been cut by the same restriction enzyme or by enzymes that generate compatible ends. Some restriction enzymes cleave DNA to produce blunt ends. Blunt ended molecules can also be joined to produce a recombinant DNA molecule -Once DNA has been cleaved by a restriction enzyme, the DNA can be cloned into a vector that has also been cut by the same restriction enzyme. The genomic DNA and vector DNA are mixed, the sticky ends anneal the genomic DNA to the vector, and the enzyme DNA ligase restores the phospdiester backbone of the two DNA strands, covalently attaching the two pieces together. The vector and inserts can be transformed into a host cell. -Cloning vectors contain many of the same features: a multiple cloning site, which a collection of many different kinds of restriction sites; an appropriate origin or replication, so the plasmid can replicate in the particular host cell chosen; and a selectable marker, which allows for the rare, transformed cells to preferentially survive certain conditions relative to their untransformed neighbors. Common vectors include, plasmids, cosmids, YACs and BACs, each with their own advances and disadvantages -once a genomic library is completed, the DNA within that library can be sequenced. One popular method of DNA sequencing uses dideoynucleotides to terminate chain extension in a modified version of DNA replication. The terminated fragments are detectable because the individual ddNTPs have a colored dye linked to them. The dye allows the fragments to be visualized and provides the information on which ddNTP terminated the fragment. A new sequencing technique, called pyrosequencing, directly detects the identity of each nucleotide as it incorporated into the growing DNA strand, so no chain termination is needed -there are a number of different approaches to sequencing whole genomes. The technique now prevalently used is the whole-genome shotgun approach. In this approach, the genome first is broken down into random, o
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