JC 15 Aug1 by B4Z4B9U2

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									                                   DNA
       DNA                                       mRNA
                              5’
                               process     3’
             ribosome
                                                mature
mRNA
                                                mRNA
                        cap
                         5’                       3’
        proteins                                 tail

                                proteins
Prokaryote
                                Eukaryote
     Technique involving the insertion of a
fragment of foreign DNA into a vector
capable of replicating autonomously in a
host cell (usually Escherichia coli). Growing
the host cell allows the production of
multiple copies of the inserted DNA for use
in a variety of purposes.
 Foreign DNA

 Host organism

 Vector DNA for cloning

 Means of inserting foreign DNA into the vector

 Method of placing the in vitro modified DNA
     into the host cell

 Methods for selecting and/or screening cells that
     carry the inserted foreign DNA
Restriction Endonucleases
    Type I – Long recognition sequences and random to cleavage

    Type II – Tetra, Penta and Hexa recognition sequences

    Type III – Similar to type I but specific to clevages
Polymerases
    DNA Polymerase – catalyzes the polymerization of
          deoxyribonucleotides along the template strand

    DNA-dependent RNA Polymerase
Nucleases
    Enzymes capable of cleaving the phosphodiester bonds
         between nucleotide subunits of nucleic acids
Other Modifying Enzymes

  Ligases
            forms phosphodiester bonds to join two pieces of DNA
            utilizes ATP in the presence of Mg++
            T4 DNA ligase for “blunt”or “cohesive” ends
            E.coli DNA ligase for “blunt” ends
  Kinases
            transfers phosphate groups from donor molecules
            phosphorylase
  Phosphatases
            catalyzes the removal of 5’-phosphate residues
 Foreign DNA
    Genomic DNA

    Complementary DNA (cDNA)

    Synthetic DNA

         Chemical Synthesis

         Polymerase Chain Reaction (PCR)
 Vector DNA
         DNA molecule that functions as a “molecular carrier”
that carry the DNA of interest into the host cell & facilitates
its replication.

Plasmids – used in cloning small segments of DNA (10-15 kb)

Bacteriophage – used in cloning larger segments of DNA
              (~20 kb)

Cosmids – plasmids containing DNA sequences (cos) from
              bacteriophage λ used to clone larger fragments
              of up to 45 Kb
• small circular dsDNA that autonomously replicates
       apart from the chromosome of the host cell

• “molecular parasites”

• carry one or more genes some of which confer
       resistance to certain antibiotics

• origin of replication (ORI) --- a region of DNA that
       allows multiplication of the plasmid within the
       host

• plasmid replication: stringent or relaxed
Desirable properties of plasmids:

       small size

       known DNA sequence

       high copy number

       a selectable marker

       a second selectable gene

       large number of unique restriction sites
• viruses that infect bacteria

• known dsDNA sequence of ~ 50 kb

• linear double-stranded molecule with
       single-stranded complementary ends

• cohesive termini (cos region)




                           http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif
Desirable properties of λ phage:

      • can accept large pieces of foreign DNA

      • tremendous improvement over the years

      • can be reconstituted in vitro
• modified plasmids containing cos sequences

• carry an ORI & an antibiotic resistance marker

• can accommodate ~35 to 45 kb of foreign DNA

• can be propagated as plasmids

• can be introduced into host by standard
      procedures

• chief technical problems occur when used for
       library construction
? Cloning or Expression

? Single or Library gene construct

? Size of foreign gene

? Restriction size

? Regulatory sequences

? Signal sequences

? Tag sequences
http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif
http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif
 Means of inserting foreign DNA into the vector
Ligation of the DNA into the linearized vector




                                          http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/ligation.gif

Requirements for a ligation reaction:
 • two or more fragments of DNA (blunt/cohesive)
  ng of vector x kb size of insert x insert:vector molar ratio = ng of insert
         kb size of vector

 • buffer containing ATP

 • T4 DNA ligase
 Method of placing the in vitro modified DNA
     into the host cell


Transformation into the host cell

  • bacterial cells take up naked DNA molecules

  • cells are made “competent”

  • cells treated with ice-cold CaCl2 then heat-shocked

  • efficiency of 107 to 108 transformed colonies/μg DNA

  • maximum transformation frequency of 10-3
Electroporation of the DNA into the host cell




                                                http://bme.pe.u-tokyo.ac.jp/research/ep/img/electroporation.jpg
• “electric field-mediated membrane
        permeabilization”

• high strength electric field in the
        presence of DNA

• protocols differ for various species

• efficiencies of 109 per μg DNA (3 kb)
         & 106 (136 kb)
Conjugation
 • natural transmission from donor to recipient

 • host cell that is not readily transformed

 • form cell to cell junctions


Transfection of the DNA
 • DNA is packaged in vitro into phage particles

 • phages are allowed to infect bacterial cells

 • term also used in DNA transfer to eukaryotic cells

 • DNA is transiently expressed
 Escherichai coli
        Well Characterized genetic system
        Easily to be manipulated for maximum gene expression
        Release of exdotoxin
        Excrete very few proteins
 Bacillus subtilis
        Secretes many proteins
        Non- pathogenic strian
        Transcribe only genes from closed relative genus
        Vector instability
 Streptomeces sp.
        Already being used at large scale
        Non- pathogenic strian
        Transcribe genes from wide range
        Less understood about genetic system
 Methods for selecting and/or screening cells that
     carry the inserted foreign DNA


Selection refers to application of conditions that
      favors the growth of cells or phages that
      carry the vector or vector and insert.


      • antibiotic resistance

      • nutrient requirements

      • plaque formation
Screening allows all cells to grow, but tests the
      resulting clones for the presence of the insert
      in the vector.

      • restriction analysis

      • antibiotic resistance/sensitivity

      • nutrient requirements

      • plaque type

      • alpha complementation

      • specific (PCR, hybridization, product assay)
• vector carrying lacZ gene – encodes for N-terminus of
        β-galactosidase (α-fragment)

• host chromosome carrying C-terminal of β-galactosidase
        (ω-fragment)

• IPTG (isopropyl-[beta]-D-thiogalactopyranoside) – inducer

• X-Gal – substrate for the enzyme

• cloning sites within the LacZ gene

• disruption of gene by insertion

       of the foreign DNA

• blue – functional protein

• white – non-functional protein          http://www.eppendorfna.com/applications/images/gel_cleanup1.jpg
• vector carrying
two or more
antibiotic
resistance genes

• cloning sites
within the one of
antibiotic
resistance gene

• disruption of gene
by insertion of the
foreign DNA
• Southern hybridization – Detection of DNA by DNA probe

• Nortern hybridization – Detection of RNA by DNA probe

• Western hybridization – Detection of Protein by antibody probe
 Isolation of desired genes                 DNA vector isolation



Restriction enzyme digestion             Restriction enzyme digestion


   Digested DNA fragment                     Digested DNA vector


                              Ligation


                      Recombinant plasmid


                 Transformation into host cells


            Selection/Screening of recombinant clones
Maxam-Gilbert's Method:               Chemical        Destroy → Cleavage
                                       method
                                    32P      ATCGATCG              AT
32P     ATCGATCGAT
                                    32P
                                             ATCG         ATCGAT
                                                          Non-radioactive
      Specific Reaction to G                                (invisible)
                                              Destroy → Cleavage


Sanger's Method:        Biosynthetic method           Keep on going
                     Terminator          ATCGA
             A,T,C,G     A               TAGCTAGCTA
                         32P
        ATCG                          or Producing various fragments
        TAGCTAGCTA                             STOP
                                                    Terminated
           Template                      ATCG
                                                    32P
                                                      A
                                             TAGCTAGCTA
DNA fragments having a difference
of one nucleotide can be separated     Polyacrylamide Gel Electrophoresis
on gel electrophoresis                      T      A     G     C
 32P
          ATCGATCGAT
 32P
          ATCGATCGA
 32P      ATCGATCG
 32P      ATCGATC
 32P      ATCGAT
 32P
          ATCGA                                                       G
 32P      ATCG                                                        C
 32P      ATC
                                                                      T
 32P
          AT
 32P
          A                                                           A
       But these bands can’t tell us   If those band with the same
       the identity of the terminal    terminal nucleotide can be
       nucleotides                     grouped, then it is possible
                                       to read the whole sequence
 Open reading frame

 Aminoacid sequences

 Hydrophobic profile

 Homology with other proteins

 Inverted repeated sequences

 Search for promoter region(s) or transcription site
 Genetically engineered E.coli to produce antigen
for develop to vaccine or diagnostic kit test

 Genetically engineered E.coli to produce antibody
for therapeutics or diagnostic kit test

 Genetically engineered        E.coli   to     produce
hormones for therapetics
?

								
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