Cloning

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					Cloning
 Guanqun Yuan
 12-13-2004
 R.A.Scott Group Meeting
Outline
 What is cloning?
 How to do cloning?
 Validation
 Application
What is Cloning?
   Generating identical copies of organisms, cells,
    or replicating nucleic acid sequences from
    organisms
   involving human intervention.
   Giving rise to new generation
   Dolly (the sheep) is a clone, but a natural
    identical twin is not a clone.
   DNA sequence amplified by growth is a clone,
    Identical DNA molecules produced in vitro (a
    PCR rxn) is not a clone.
Outline
 What is cloning?
 How to do cloning?
 Validation
 Application
      How?
ORI                                                                                                         Cells that do
              Amp R.
                                                                                                            not take up
                                     ORI               Amp R.                                             plasmids die on
                                                                                                          ampicillin plates


  Plasmid vector    Enzymatically                        Mix E.coli cells with
                   insert DNA into         Recombinant plasmids in presence of
      +             plasmid vector           plasmid   CaCl2 Culture on nutrient
                                                                                                           Transformed
                                                                                                            E.coli cell
                                                        agar plates containing                               survives
  DNA fragment                                                ampicillin
   to be cloned                                                                     Bacterial
                                                                                  chromosome

                                                                                                Independent
                                                                                                  plasmid
                                                                                                 replication
                                                                      Cell
                                                                 multiplication
Composition
   Vectors
      Plasmids, or phage   Plasmid vector


   The cell
      E.coli, yeast
   Inserted sequence
                             DNA fragment
                              to be cloned
Vectors
  The substance that can serve as carriers to allow
  replication of recombinant DNAs.
 Plasmids
 Phage λ
 Plasmid phage hybrids
                                                   MCS

Plasmids                                           Ab. Resis
                                         ORI

ds circles of DNA that can replicate autonomously.
Three features of the plasmid cloning vectors:
 Multiple cloning site. The place where foreign DNA
  fragments can be inserted.
 An origin of replication. The replication origin is a specific
  DNA sequence of 50-100 base pairs that must be
  present in a plasmid for it to replicate. Host-cell enzymes
  bind to ORI, initiating replication of the circular DNA.
 A gene specifying resistance to an Antibiotic. This
  permits selective growth of the host cell.
   Most often used: Resistance to ampicillin, penicillin,
  tetracycline, kanamycin, and chloramphenicol.
Phage λ
   A phage λ virion has a head, which
    contains the viral DNA genome, and a
    tail, which functions in infecting E.coli
    host cells.                                 Viral
                                                Genome

   Advantages over plasmids: They infects
    cells much more efficiently than
    plasmids transform cells. The yield of
    clones with vectors usually higher.
   Because of its efficiency, phage λ is
    often used in library construction.
The Cell
E.coli:
   Normal E. coli cells cannot take up plasmid DNA from
    the medium. Exposure to high concentration of certain
    divalent cations, CaCl2, makes a small fraction of cells
    permeable to foreign DNA.
   Each component cell incorporates a single plasmid DNA
    molecule, which carries an antibiotic-resistance gene.
    When the cells are treated wit antibiotics on plates, only
    a few of the transformed cells containing the antibiotics-
    resistance gene on the plasmid vector will survive.
Inserted Sequence
   Source of Nucleic acid to be cloned:
    -DNA directly from organism
    -DNA synthesized or amplified in vitro (cDNA or PCR
    reactions).
    -Previously cloned DNA. Generally a specific sequence.
   Quality of DNA can be crucial
    -Its purity, being free of contaminants
    -its size is crucial when cloning very large pieces
Two Important Enzymes
   Restriction Enzymes:
    cuts the DNA from any organism at specific sequences
    of a few nucleotides, generating a reproducible set of
    fragments.
   DNA Ligases:
    insert DNA restriction fragments into replicating DNA
    molecules producing recombinant DNA.
Mechanism
  Restriction Enzyme: EcoRI
5`      GAATT C                 3`                 5`            G      AATT C                3`
3`      C T T AA G              5`                 3`            C T T AA       G             5`
                     Cleavage
                                                                            Sticky ends
  DNA Ligases                                               OH
  5`         OH                           5`                A A T T              3`        Unpaired
  3`       T T A A      P                               P               P              +   B and C
                                          3`                T T A A              5`
       +                                                              OH
                             Complementary
  5`         OH
A                            ends base-pair
  3`       T T A A      P
                                                            DNA
                                                            ligases
B 5`        OH
  3`       C G   P
                                              5`            A A T T               3`
C 5`         OH
  3`       T A C G       P                    3`            T T A A               5`
Outline
 What is cloning?
 How to do cloning?
 Validation
 Application
Validation
Because introducing DNA into an organism is
  usually not very efficient, we need to do
  validation.
 Selection- A technique that isolates only a
  particular type of cell or organism.
 Screen- A technique that allows identifying a
  particular type of cell or organism but does not
  isolate it from other types.
 Selection
   pBR322
                     Amp. R


            Tet. R                       Tet. R
EcoRI                         Amp. R




                                                  Cell




                                Ampicillin
    Screen
                                                        Cell
   pBR322

                          EcoRI   EcoRI                           Tetracycline
        Amp R.                                 Tet. R
EcoRI            Tet. R




                                                               Replica
                                                               plating
                                                               process
                                          Add Amp.




                                                               The cells we want
   Other validation methods
                 About 100 bp
  Promoter       Multi. cloning site                                         U.P.
                     EcoRI                  BamHI
Ori.
                                                    SalI                              L.P.
                                                           PCR
                Ab. Resis.
         +
   EcoRI        EcoRI
                                               Restriction
       Inserted Gene
       About 500bp-5kb                         Enzyme
                                                                 Marker   With Prod. Without Prod.
       Marker    With Prod. Without Prod.
Sequencing
Dideoxy: (Sanger) Manual
   Primer extension reactions in four separate tubes.
   Using a dideoxy nucleotide as the chain terminator.
   Each tube contains different dideoxy nucleotide (ddATP,
    ddCTP, ddGTP, ddTTP).
   Radioactive dATP is also included in all the tubes so the
    DNA products will be radioactive.
   The results is a series of fragments of different lengths.
   Finally, autoradiography is performed to visualize the
    DNA fragments.
  Dideoxy: (Sanger) Manual
a) Primer extension reaction                        c) Electrophoresis of the Protein
                    TACTATGCCAGA                              ddA   ddC   ddG   ddT
                                                                                      T
   20-base primer        Replication with ddTTP                                       C
                                                                                      T
                    TACTATGCCAGA                                                      G
                    ATGA T                                                            G
   25-base primer
                                                                                      C
                                                                                      A
                                                                                      T
                                                                                      A
                                                                                      G
                                                                                      T
b) Product of the four reactions                                                      A

   Product of ddA rxn                               Product of ddC rxn
 Template:          TACTATGCCAGA                  Template:          TACTATGCCAGA
      (21)          A                                  (27)          ATGATAC
      (24)          ATGA                               (31)          ATGATACGGTC
      (26)          ATGATA


   Product of ddG rxn                               Product of ddA rxn
 Template:          TACTATGCCAGA                  Template:          TACTATGCCAGA
      (23)          ATG                                (22)          AT
                                                       (25)          ATGAT
      (28)          ATGATACG                           (30)          ATGATACGGT
      (29)          ATGATACGG
                                                       (32)          ATGATACGGTCT
Sequencing
Dideoxy: (Sanger) automated
   The “manual” sequencing technique is powerful but slow,
    thus Rapid automated sequencing methods are required.
   Still based on the procedure using dideoxy nucleotides,
    but tagged with a different fluorescent molecule, so the
    product from each tube will emit a different color
    fluorescence when excited by light
   After extension reaction and chain termination, all 4
    solutions are mixed and electrophoresed together in the
    same lane on gel analyzed by laser beam
   The color of the fluorescent light emitted from each
    oligonucleotide is detected electronically
Outline
 What is cloning?
 How to do cloning?
 Validation
 Application
Application

Expression
Library
Expression

Why?
You want the cloned gene to make its product, normally a
  protein.
 Identifying gene from library requires expression.
 To overproduce the protein and purify it.
 For in vivo studies of the protein.
Expression
Expression Vectors:
Vectors that can yield the protein products of the cloned
  genes.
Two elements that are required for active gene
  expression: a strong promoter and a ribosome binding
  site near an initiating ATG codon.
The main function of an expression vector is to yield the
  product of a gene, therefore a strong promoter is
  necessary. The more mRNA is produced, the more
  protein product is made.
Inducible Expression Vectors
   Protein produced in a large quantity in bacteria can be
    toxic, so it is advantageous to keep a cloned gene
    repressed before expressing it.
   Solution: keep the cloned gene turned off by placing it
    downstream of an inducible promoter that can be turned
    off.
   IPTG strongly induce lac promoter
Expression
Expression Vector that Produce Fusion Proteins
 Fusion proteins: Gene (or part of a gene) for one
  protein fused to part or all of a gene for a second protein.
Major uses for generating fusion proteins:
 The „tag‟ of the fusion protein can greatly aid biochemical
  purification. If the tag binds a particular substance, a
  column prepared containing that bound substance can
  be used to purify the tagged protein from virtually all
  other proteins.
           (His)6
     ATG
                    MCS   Oligohistidine regions like this have a
                          high affinity for metals like nickel, so
                          proteins that have such regions can be
                          purified using nickel affinity
                          chromatography
Fusion Protein
   The „tag‟ can serve as a convenient way for
    identification of the tagged protein in cells or extracts.
    For example, a short peptides can be sufficient to use as
    a tag for antibody binding. In this way, a common
    antibody can be used, eliminating the need to develop a
    novel reagent specific to the protein of interest.
   The „tag‟ can be used as a surrogate in the
    quantification of the tagged protein. Again, a routine
    assay of the activity of the tag can be used to monitor
    amounts of a protein of interest that may have no means
    of assay otherwise.
Library Construction
A library is a collection of different cloned DNAs
  from a single source that are present in different
  copies of a particular cloning vector.

   Genomic library – for genome sequencing
   cDNA library – derived from mRNA of a
    particular tissue, for isolating specific genes
Library Construction
The principle of library construction is basically quite simple.
 Cut a DNA vector at a unique restriction site and ligate
  into it the DNA that you want to make a library out of.
 If you want a library of human genomic DNA, you use
  fragmented human DNA.
 The ligation mix is not yet considered the library. The
  library comes after the generation of E. coli cells carrying
  the cloned DNA.
 To generate a library with a million clones for example,
  you need to recover a million independent colonies
  carrying plasmids or a million independent phage
  plaques. By pooling together all the independent clones
  you get the library.
Library Construction
Though simple in principle, libraries are difficult to make well.
 Partly this is just a matter of scale. While in routine
  cloning, you generally just need to recover a single type of
  clone, a library has to generate very large numbers of
  independent DNA inserts.
 While used pretty frequently, libraries are seldom made.
  Few people have much experience.
 The best advice for making a library is to not do it unless
  you really have to. Get a library from someone else that
  has already made one. Some are commercially available.
Thanks
Dr. Robert A. Scott
All the members in the group
Xiaoming Wang
Reference
 Manuscript of Course Genetics 8920
 Molecular Biology
                    Robert F. Weaver
 Molecular Cell Biology
      Lodish, Berk, Zipursky, Matsudaira,
      Baltimore, Darnell

				
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