DNA_Technologyfinal

DNA_Technologyfinal - DNA Technology Molecular Biologists...

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DNA Technology
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Molecular Biologists had a problem The molecular biologist studying a particular gene faces challenges: Naturally occurring DNA molecules are very long Moreover, genes may occupy only a small proportion of the chromosomal DNA, the rest being noncoding nucleotide sequences. (A single human gene, for example, might constitute only 1/100,000 of a chromosomal DNA molecule) The distinctions between a gene and the surrounding DNA are subtle
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Genes and bacteria What can gene cloning do?
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Gene cloning is possible because of Restriction Enzymes Restriction enzymes- An enzyme that cuts double-stranded DNA. AKA -restriction endonuclease
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Figure 8-31 Molecular Biology of the Cell (© Garland Science 2008)
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Transformation http://www.dnai.org/b/index.html
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Using a Plasmid Vector X-gal is used to indicate whether a bacterium expresses the β-galactosidase enzyme, which is encoded by the lacZ gene, in a technique called blue/white screening.
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Using a Phage Vector
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Genomic Library Genomic libraries. A genomic library is a collection of many bacterial or phage clones, each containing copies of a particular DNA segment from a foreign genome.
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cDNA and cDNA Libraries Complementary DNA (cDNA)- A DNA molecule made mRNA as a template and the enzyme reverse transcriptase. cDNA library- A limited gene library using complementary DNA. The library includes only the genes that were transcribed in the cells examined.
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Cloning Large Amounts of DNA Plasmids are good for small pieces of DNA Use artificial chromosomes for larger molecular clones (required for sequencing) All artificial chromosomes contain an origin of replication, a centromere sequence, and the foreign DNA Types of artificial chromosomes YAC’s- first artificial chromosome, has a tendency to rearrange, or loose portions by deletion HAC’s- introduces genes into cultured cells, they are circular and can sometimes segregate correctly, linear HAC construction is still not possible BAC’s- most commonly used
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Figure 8-45b Molecular Biology of the Cell (© Garland Science 2008) Polymerase Chain Reaction (PCR)
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Identifying Clones Carrying a Gene of Interest Nucleic acid hybridization- Base pairing between a gene and a complementary sequence on another nucleic
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This note was uploaded on 11/16/2011 for the course BIO 102 taught by Professor Brey during the Fall '08 term at Rutgers.

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DNA_Technologyfinal - DNA Technology Molecular Biologists...

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