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    Recombinant DNA is DNA derived from at least two genetic sources. DNA is manipulated through isolation, digestion with restriction enzymes, and cloning of DNA fragments into vectors. Vectors have attributes like origins of replication, multiple cloning sites, selection markers, and the ability to be maintained in host cells. Recombinant plasmids are identified through transformation, selection, and characterization of fragments by gel electrophoresis and restriction mapping. DNA and RNA clones of interest are identified through hybridization techniques like Southern blotting, Northern blotting, and sequencing. Microarrays allow analysis of global gene expression patterns through hybridization of fluorescently labeled cDNA or cRNA to arrays of oligonucleotides representing genes.

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    Recombinant DNA is DNA derived from at least two genetic sources. DNA is manipulated through isolation, digestion with restriction enzymes, and cloning of DNA fragments into vectors. Vectors have attributes like origins of replication, multiple cloning sites, selection markers, and the ability to be maintained in host cells. Recombinant plasmids are identified through transformation, selection, and characterization of fragments by gel electrophoresis and restriction mapping. DNA and RNA clones of interest are identified through hybridization techniques like Southern blotting, Northern blotting, and sequencing. Microarrays allow analysis of global gene expression patterns through hybridization of fluorescently labeled cDNA or cRNA to arrays of oligonucleotides representing genes.

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    8 views14 pages

    Lecture 9 BB1035 C15

    Uploaded by

    Josh Fuller
    Recombinant DNA is DNA derived from at least two genetic sources. DNA is manipulated through isolation, digestion with restriction enzymes, and cloning of DNA fragments into vectors. Vectors have attributes like origins of replication, multiple cloning sites, selection markers, and the ability to be maintained in host cells. Recombinant plasmids are identified through transformation, selection, and characterization of fragments by gel electrophoresis and restriction mapping. DNA and RNA clones of interest are identified through hybridization techniques like Southern blotting, Northern blotting, and sequencing. Microarrays allow analysis of global gene expression patterns through hybridization of fluorescently labeled cDNA or cRNA to arrays of oligonucleotides representing genes.

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    of 14

    Lecture 9 Recombinant DNA

    Recombinant DNA & Biotechnology


    What is recombinant DNA?
    DNA molecule made in the laboratory derived from at
    least two genetic sources.
    How is DNA manipulated?
    isolate DNA (from)
    - properties of chromosomal DNA (large, acidic)
    - properties of vector DNA
    digest DNA
    - shearing (intentional, unintentional)
    - restriction enzymes
    GAATTC
    CTTAAG
    recognition sequences = specific (Why?)
    palindromic: same sequence antiparallel

    Lecture 9 Recombinant DNA


    How is DNA manipulated?
    staggered vs blunt (sticky or cohesive)

    Lecture 9 Recombinant DNA


    directed vs undirected
    EcoRI

    BamH1
    EcoRI

    BamH1

    BamH1

    BamH1

    BamH1

    BamH1

    BamH1
    BamH1

    EcoRI

    BamH1

    Lecture 9 Recombinant DNA


    How is DNA manipulated?

    isolate DNA (from)


    - properties of chromosomal DNA (large, acidic)
    - properties of vector DNA
    clone fragments into vector
    - attributes

    1. ori
    2. mcs/polylinker
    3. selection
    a. vector
    b. insert
    4. small size
    5. high copy #
    6. host range
    7. size of insert
    8. Growth
    9. Purification
    10. Stability
    11. Storage
    12. expression
    Vectors Plasmids, YACS, cosmids,
    TI plasmid T DNA, viruses

    Lecture 9 Recombinant DNA


    Identifying recombinant plasmids
    transformation/selection/transgenic

    Lecture 9 Recombinant DNA


    Useful Markers
    selectable
    screenable

    Libraries

    Lecture 9 Recombinant DNA


    Libraries
    isolate DNA (from)
    - genomic

    Genomic
    - noncoding
    - digestion
    partial vs complete
    - size/representation:
    # of clones x insert size
    = 10X genome size
    - stability
    repetitive DNA

    mRNA (how do we isolate??)


    1. location? (cytoplasm)
    2. processing? (poly A tail)
    cDNA
    - what cells, tissue, organ,
    developmental stage?

    Oligo dT

    - abundance
    - how to clone

    Reverse transcriptase

    RNaseH
    DNA pol

    Lecture 9 Recombinant DNA


    Identifying recombinant plasmids
    characterize fragments
    - gel electrophoresis (DNA properties)
    - restriction mapping
    transform

    Large

    Identify correct plasmid

    Small

    Lecture 9 Recombinant DNA


    Identifying recombinant plasmids
    How do we identify DNA/RNA clone of interest??
    Complementarity!!!
    - Southern Blot
    - Northern Blot
    - sequence analysis

    - Run DNA/RNA
    - transfer to filter
    - hybridize probe to filter
    - detect signal

    Lecture 9 Recombinant DNA


    Southern analysis:
    RI

    RI
    5kb
    1

    probe

    RI

    12.5kb
    2

    1
    12.5kb

    DNA Gel
    5kb

    2
    12.5kb

    Southern blot

    Lecture 7 Recombinant DNA


    How do we identify DNA/RNA clone of interest?
    Northern:
    Northern analysis:
    - isolate mRNA (from)
    probe
    - electrophoresis (ssRNA vs dsDNA
    structure)
    - transfer of mRNA to filter (wicking)
    - incubate w/ labeled DNA (ss probe)
    - wash
    1
    2
    - develop (radioactive vs nonradioactive)
    12.5kb

    5kb
    Region of genome is transcribed = gene!

    Lecture 9 Recombinant DNA


    DNA manipulation hybridization
    Gene analysis on a global scale - microarrays
    1. Array DNA sequences oligonucleotides (what defines a gene)
    2. Isolate mRNA - distinct samples
    3. Label samples distinct
    fluorescent dyes
    4. Hybridize to microarray
    5. Detect fluorescent signals
    expression ratio

    Yellow:
    Red:
    Green:

    Lecture 9 Recombinant DNA

    wild type
    oncogenic

    Lecture 9 Recombinant DNA


    Gene analysis on a global scale - microarrays
    6. Uses ?
    - temporal changes in gene expression during development
    - different tissues
    - +/- drug, hormone etc
    - Wild type vs disease (define diagnostic profiles)
    - Genotyping

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