Scribd Logo
Upload

Welcome to Scribd!

  • 26 views

    Biol3451 Ch19 Lect

    Uploaded by

    maniiiiiiii
    Recombinant DNA technology allows for the isolation and manipulation of genes. Key steps include using restriction enzymes to cut DNA at specific sequences, joining DNA fragments into cloning vectors, transferring the vectors into host cells, and isolating the recombinant DNA. This technology revolutionized research by enabling the production of large amounts of isolated genes and proteins.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd

    Biol3451 Ch19 Lect

    Uploaded by

    maniiiiiiii
    26 views37 pages
    Recombinant DNA technology allows for the isolation and manipulation of genes. Key steps include using restriction enzymes to cut DNA at specific sequences, joining DNA fragments into cloning vectors, transferring the vectors into host cells, and isolating the recombinant DNA. This technology revolutionized research by enabling the production of large amounts of isolated genes and proteins.

    Copyright

    © Attribution Non-Commercial (BY-NC)

    Available Formats

    PPT, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Recombinant DNA technology allows for the isolation and manipulation of genes. Key steps include using restriction enzymes to cut DNA at specific sequences, joining DNA fragments into cloning vectors, transferring the vectors into host cells, and isolating the recombinant DNA. This technology revolutionized research by enabling the production of large amounts of isolated genes and proteins.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    26 views37 pages

    Biol3451 Ch19 Lect

    Uploaded by

    maniiiiiiii
    Recombinant DNA technology allows for the isolation and manipulation of genes. Key steps include using restriction enzymes to cut DNA at specific sequences, joining DNA fragments into cloning vectors, transferring the vectors into host cells, and isolating the recombinant DNA. This technology revolutionized research by enabling the production of large amounts of isolated genes and proteins.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 37

    Recombinant DNA

    Technology

    Chapter 19
    Recombinant DNA Technology
    • 1971 paper by Kathleen Dana and Daniel
    Nathans described isolation of enzyme that
    cleaved DNA at specific sequences
    • 1978 Nobel Prize to Nathans, Smith and
    Arber for restriction endonuclease
    discovery
    Cloning DNA Molecules
    • Purify DNA from desired source
    • Cut DNA with restriction endonuclease
    • Join fragments to cloning vectors cleaved with
    compatible restriction endonuclease to create
    recombinant DNA molecules
    • Transfer recombinant molecules to host cells
    • Isolate DNA from individual clones of
    transformed host cells
    • Do as you please with the isolated DNA
    segments…
    Restriction Endonucleases
    • Hundreds now identified
    • Host cell defense against invading DNAs
    • Cleave DNA at or near a specific recognition
    sequence
    – Creates “restriction fragments”
    – Recognition sites can be from 4 to 8+ base pairs and
    are commonly palindromes
    • First one isolated was EcoRI from E. coli
    • Often produce “sticky ends”
    Restriction
    Endonucleases
    DNA Cleavage By EcoRI
    Recombinant DNA Molecules
    Cloning Vectors
    • Plasmids
    • Phage
    • Cosmids
    • BACs
    • YACs
    Plasmids
    • Circular independent
    replicons, origin of
    replication (ori)
    • Generally encode useful
    but not essential genes
    – E.g. antibiotic resistance or
    catabolic pathways
    • Allow cloning fragments
    up to about 10 kbp
    • Selectable markers
    • Multiple cloning sites
    Plasmid Cloning Vectors

    • As small as possible
    with minimum
    restriction endonuclease
    cutting sites in genes
    • ori
    • Selectable marker(s)
    • Multiple cloning site
    (MCS)
    • Reporter function useful
    • Plasmid has portion of lacZ
    gene flanking MCS lacZ
    • Host strain has lacZ gene Complementation
    missing this seqeunce
    • Expression of two
    components still yields a
    functional LacZ
    – Like it has 2 subunits
    • Cloning into MCS
    eliminates
    complementation
    • X-gal in media allows for
    blue/white selection
    Bacteriophage Vectors
    • Commonly based upon
    phage
    • Most internal genes
    deleted
    • Insert DNA into middle
    region (up to10-15 kb)
    • Package
    • Infect host cells with
    integrated helper phage
    to provide missing
    protein-encoding genes
    Cosmids
    • Plasmid with phage
    packaging sequence
    (cos)
    • Can clone up to 50 kb
    • Packaged into  particles
    and injected into host
    cells
    • Circularizes in cell and
    continues as a large
    plasmid
    BACs

    • Bacterial artificial
    chromosomes
    • Can clone up to 200+ kb
    DNA fragments
    • Based upon F plasmid
    • Origin, selectable marker,
    promoters to expressed
    cloned genes
    YACs

    • Yeast artificial
    chromosomes
    • Have centromere,
    telomeres and an
    origin of replication,
    plus selectable
    markers
    • Cloned segments of
    250 kb
    Expression
    Vectors
    • Also include
    regulatable high level
    expression promoter
    – T7 phage promoter
    – lac operator
    – lac repressor gene
    First Prokaryotic Host Cells
    • First clones introduced into strains of E. coli K12
    • Protocol
    – Isolate target DNA
    – Cut with RE
    – Ligate to vector
    – Transform host cells
    – Plate on antibiotic-containing medium
    – Identify recombinant plasmids
    – Identify/characterize specific clones
    Cloning into Plasmids
    Expression of Recombinant
    Genes in Eukaryotes

    • Expression is sometimes desirable in eukaryotic cells


    – Especially if post-translational modifications are important
    or study simply requires it
    Eukaryotic Expression Vectors
    • Generally similar to prokaryotic ones
    – Commonly start with prokaryotic vector
    • Construction done in E. coli
    • Shuttle vector
    – Origin
    – MCS
    – Selectable marker
    – High expression regulatable promoter
    – But also generally an intron included
    • Especially for cDNA clones
    Cloning into Plant Cells

    • Vectors based upon


    Ti plasmid
    – Derived from
    Agrobacterium
    tumifaciens plasmid
    Cloning into Mammalian Hosts

    • Especially for model systems


    • DNA integrates into a host cell
    chromosome
    – Random vs. site-directed
    • Transgenics
    – Models for diseases
    – “Improved” individuals???
    Transgenic Mammals
    Polymerase Chain Reaction
    • PCR
    • 1993Nobel Prize for Kary Mullis
    – Proposed in 1986
    • Can provide millions of fold amplification of
    a DNA fragment or sequence
    • Needle in haystack
    • Revolutionized molecular
    biology/genetics/forensics/and everything
    – Day earth changed…
    PCR Procedure
    • Denature DNA
    • Add primers, thermostabile
    DNAP, dNTPs
    • Extension
    • Denaturation
    • Bind primers
    • Extension
    • Repeat last 3 steps thirty times
    Chromosome
    Sorting
    • Important for early portion
    of human genome project
    – Simplified sequencing effort
    – Fluorescent tags on specific
    chromosomes
    Pulse Field Gel Electrophoresis

    • For very large DNA


    molecules
    – To left are intact yeast
    chromosomes
    • Electric field is
    pulsed/changed after
    very short intervals
    cDNAs
    • Copies of mRNAs in DNA
    – By reverse transcriptase
    • Needed to analyze genes and also to express
    eukaryotic genes in prokaryotic systems
    – Introns…
    • cDNA libraries
    cDNA Synthesis
    • Oligo T primer
    • Reverse transcriptase and
    deoxyribonucleotides
    • RNaseH
    • DNAP I makes second
    strand using the hairpin
    created by reverse
    transcriptase as a primer
    • Hairpin cleave by S1
    nuclease
    Library Screening
    • General approach used for
    both genomic and cDNA
    libraries
    – DNA from colonies/plaques
    – transferred to membrane
    – Denature
    – Hybridize with probe
    – Detect probe binding
    • Method depends upon
    labeling of probe
    – Isolate specific probe-
    binding clones and
    culture
    Restriction
    Maps
    • Useful but now
    most commonly
    generated by
    computer from
    actual DNA
    sequence
    Southern
    Transfer
    • Developed by E. M.
    Southern
    • Method for
    transferring DNA
    from a gel to a
    membrane
    • Described to right
    • Also Western and
    Northern blots
    – Proteins and RNA
    Hybridization

    • Binding of a probe (generally RNA or DNA) to a


    nucleic acid in a gel or more commonly bound to a
    membrane
    DNA
    Sequencing

    • Sanger
    • Method
    described
    to right
    Chain Termination DNA
    Sequencing

    • Autoradiograph of results
    from Sanger
    dideoxyribonucleotide chain
    termination method
    • Sequence is read from bottom
    to top
    Fluorescent
    Dyes
    • DNA sequencing with
    fluorescent dyes
    attached to chain
    terminator
    dideoxyribonucleotides
    • Allows for automated
    DNA sequencing
    Electropherogram

    You might also like