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Chapter 18

BIOL 1107 Chapter Notes - Chapter 18: Genetically Modified Organism, Cloning Vector, Dna Ligase


Department
Biology
Course Code
BIOL 1107
Professor
Thomas Abbot
Chapter
18

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18.1 Key DNA Tech for Making Genetically Altered Organisms
βž” Genetically altered organism: organism that has its genome altered to change a genetic trait
βž” Gene cloning: producing many identical copies of a gene in a host cell
β—† Clone: line of genetically identical cells or individuals derived from a single ancestor
β—† Applications include
● Gene therapy to correct/ treat genetic diseases like sickle-cell anemia
● Production of pharmaceuticals such as insulin to treat diabetes
βž” Genetically modified organism (GMO): genetically altered organism whose genome has been
engineered to introduce/change a genetically controlled trait
βž” Most genetic alterations
β—† Modify a gene to change function, delete a gene, add a gene/regulatory sequence from another
organism
β—† All alter genotype and phenotype
β—† Involve cutting DNA (using nucleases), joining DNA (using DNA ligase), making many copies
of the DNA (using DNA polymerases)
βž” Recombinant DNA: DNA fragments from 2 or more sources that have been joined together to form a
single molecule
β—† Introduced into a bacterium to produce a genetically altered bacterium
● Bacterium is cultured, cell grows and divides and recombinant DNA replicates
βž” Restriction endonucleases (restriction enzymes): bacterial enzymes that can be used to generate DNA
molecules that can be joined to produce recombinant DNA molecules
β—† Recognize restriction sites, specific DNA sequences that are typically 4-8 bp long, and cut DNA
at specific locations
β—† Name refers to regular function, enzymes defend against viral attack by breaking down DNA
molecules of infecting viruses
● Bacterium protects its own DNA by chemically modifying bases in the restriction sites so
the enzyme can’t recognize them
βž” Restriction fragments: DNA fragments produced by cutting a long DNA molecule with a restriction
enzyme
βž” Most restriction sites are symmetrical, nucleotides read 5’ β†’ 3’ on one strand is same as sequence
read 5’ β†’ 3’ on complementary strand (restriction sites are palindromes)
βž” Restriction enzymes most used in cloning cleave the sugar-phosphate backbones of DNA to produce
fragments with single-stranded ends
β—† Sticky end: end of a DNA fragment generated by digestion with a restriction enzyme, with a
single-stranded structure that can form hydrogen bonds with a complementary sticky end on any
other DNA molecule cut with the same enzyme
βž” Ligation: process of sealing 2 DNA molecules together with DNA ligase to produce a recombinant DNA
molecule
β—† Restores sequences of the 2 restriction sites
βž” Cloning vector: DNA molecule into which a DNA fragment can be inserted to form a recombinant DNA
molecule for the purpose of cloning
βž” Plasmid cloning vectors contain 2 genes that are useful in the final steps of a cloning experiment for
sorting out bacteria that have recombinant plasmids
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