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Recombinant DNA
   Technology
    BTEC3301
Recombinant DNA Technology



 Recombinant DNA technology procedures
 by which DNA from different species can
 be isolated, cut and spliced together --
 new "recombinant " molecules are then
 multiplied in quantity in populations of
 rapidly dividing cells (e.g. bacteria, yeast).
Recombinant DNA Technology



 The term gene cloning, recombinant DNA
 technology and genetic engineering may
 seems similar, however they are different
 techniques in Biotechnology and they are
 interrelated
Recombinant DNA Technology



 Human gene therapy, genetically-
 engineered crop plants and
 transgenic mice have become possible
 because of the powerful techniques
 developed to manipulate nucleic acids and
 proteins.
Recombinant DNA Technology



 In the early 1970s it became possible to
 isolate a specific piece of DNA out of the
 millions of base pairs in a typical genome.
Recombinant DNA Technology



 Currently it is relatively easy to cut out a
  specific piece of DNA, produce a large
  number of copies , determine its
  nucleotide sequence, slightly alter it and
  then as a final step transfer it back into cell
  in.
Recombinant DNA Technology



Recombinant DNA technology is based on a
        number of important things:
 Bacteria contain extrachromosomal
  molecules of DNA called plasmids
  which are circular.
Recombinant DNA Technology




   Bacteria also produce enzymes called
    restriction endonucleases that cut
    DNA molecules at specific places into
    many smaller fragments called
    restriction fragments.
Recombinant DNA Technology

Restriction Enzymes and plasmid
 There are many different kinds of
  restriction endonucleases
 Each nuclei cuts DNA at a specific site
  defined by a sequence of bases in the
  DNA called a recognition site
Recombinant DNA Technology

Restriction Enzymes and plasmid
 A restriction enzyme cuts only double-
 helical segments that contain a particular
 sequence, and it makes its incisions only
 within that sequence--known as a
 "recognition sequence".
Recombinant DNA Technology

Restriction Enzymes and plasmid
 Sticky end and blunt end are the two
 possible configurations resulting from the
 breaking of double-stranded DNA
Recombinant DNA Technology

    Restriction Enzymes and plasmid
 If two complementary strands of DNA are of
    equal length, then they will terminate in a
    blunt end, as in the following example:

   5'-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT-3'

   3'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA-5'
Recombinant DNA Technology

 Restriction Enzymes and plasmid
 However, if one strand extends beyond the
 complementary region, then the DNA is said
 to possess an overhang:

 5'-ApTpCpTpGpApCpT-3'
 3'-TpApGpApCpTpGpApCpTpApCpG-5'
Recombinant DNA Technology

 Restriction Enzymes and plasmid
 If another DNA fragment exists with a
 complementary overhang, then these two
 overhangs will tend to associate with each
 other and each strand is said to possess a
 sticky end:
Recombinant DNA Technology

    Restriction Enzymes and plasmid
   5'-ApTpCpTpGpApCpT    pGpApTpGpCpGpTpApTpGpCpT-3'

   3'-TpApGpApCpTpGpApCpTpApCpGp    CpApTpApCpGpA-5'

                         Becomes
   5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'

   3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'
Recombinant DNA Technology

Restriction Enzymes and plasmid
 Restriction Enzymes are primarily found in
  bacteria and are given abbreviations
  based on genus and species of the
  bacteria.
 One of the first restriction enzymes to be
  isolated was from EcoRI
 EcoRI is so named because it was
  isolated from Escherichia coli strain called
  RY13.
Recombinant DNA Technology
  Digestion of DNA by EcoRI to
produce cohesive ends ( Fig. 3.1):
Recombinant DNA Technology
     Creating recombinant DNA :

 The first Recombinant DNA molecules
 were made by Paul Berg at Stanford
 University in 1972.

 In 1973 Herbert Boyer and Stanley Cohen
 created the first recombinant DNA
 organisms.
Recombinant DNA Technology

Creating Recombinant DNA (Fig 3.2):
Recombinant DNA Technology
   Reading materials :Summary of
    Recombinant DNA technology
             process:
 Recombinant DNA technology requires
 DNA extraction, purification, and
 fragmentation.

 Fragmentation of DNA is done by specific
 'restriction' enzymes and is followed by
 sorting and isolation of fragments
 containing a particular gene.
Recombinant DNA Technology
    Summary of Recombinant DNA
        technology process:
 This portion of the DNA is then coupled to
 a carrier molecule.

 The hybrid DNA is introduced into a
 chosen cell for reproduction and
 synthesis.
Recombinant DNA Technology
     Transformation and Antibiotic
              Selection

 Transformation is the genetic alteration of
 a cell resulting from the introduction,
 uptake and expression of foreign DNA.
Recombinant DNA Technology
    Transformation and Antibiotic
             Selection

 There are more aggressive techniques for
 inserting foreign DNA into eukaryotic cells.
  For example, through electroporation .
   Electroporation involves applying a
    brief (milliseconds) pulse high voltage
     electricity to create tiny holes in the
 bacterial cell wall that allows DNA to enter.
Recombinant DNA Technology
 Plasmids and Antibiotic resistance

 Plasmids   were discovered in the late
 sixties, and it was quickly realized that
 they could be used to amplify a gene of
 interest.

 A plasmid containing resistance to an
 antibiotic (usually ampicillin) or
 Tetracycline, is used as a vector.
Recombinant DNA Technology
 The gene of interest (resistant to
 Ampicillin) is inserted into the vector
 plasmid and this newly constructed
 plasmid is then put into E. coli that is
 sensitive to ampicillin.( Text bk:Pg 58)

 The bacteria are then spread over a plate
 that contains ampicillin.
Recombinant DNA Technology
  Plasmids and Antibiotic resistance

 The ampicillin provides a selective
  pressure because only bacteria that have
  acquired the plasmid can grow on the
  plate.
 Those bacteria which do not acquire the
  plasmid with the inserted gene of interest
  will die.
Recombinant DNA Technology
  Plasmids and Antibiotic resistance

 As long as the bacteria grow in ampicillin,
 it will need the plasmid to survive and it
 will continually replicate it, along with the
 gene of interest that has been inserted to
 the plasmid .
Recombinant DNA Technology


 Fig 3.3
   (a).

Selecting
a Gene in
a plasmid
   and
Antibiotic
selection.
Recombinant DNA Technology
Assignment: For above procedure,

 Read Transformation of Bacterial
 cells and Antibiotic selection pg
                61.
Recombinant DNA Technology
         Human Gene cloning



 Once inside a bacterium, the plasmid
 containing the human cDNA can multiply
 to yield several dozen replicas.
Recombinant DNA Technology
Recombinant DNA Technology
       Reading materials:
 Summary of Recombinant DNA and
       Cloning (Fig. below):

 Isolation of two kinds of DNA

 Treatment of plasmid and foreign DNA
 with the same restriction enzyme

 Mixture of foreign DNA with plasmids
Recombinant DNA Technology
 Addition of DNA ligase

 Introduction of recombinant plasmid into
 bacterial cells

 Production of multiple gene copies by
 gene cloning
Recombinant DNA Technology
Summary of Recombinant DNA and
         Cloning (Fig.):
Recombinant DNA Technology

 This segment is "glued" into place using
 an enzyme called DNA ligase.

 The result is an edited, or recombinant,
 DNA molecule.
Recombinant DNA Technology
 When this recombinant plasmid DNA is
 inserted into E. coli, the cell will be able to
 process the instructions to assemble the
 amino acids for insulin production.
Recombinant DNA Technology

 More importantly, the new instructions are
  passed along to the next generation of E.
  coli cells in the process known as gene
  cloning.
 Assignment: Human gene cloning pg 63
Recombinant DNA Technology



   Fig:
Inserting
  a DNA
 sample
  into a
 Plasmid
Recombinant DNA Technology
                             References

 http://en.wikipedia.org/wiki/Restriction_enzyme
 http://web.mit.edu/esgbio/www/rdna/cloning.html
 http://faculty.plattsburgh.edu/donald.slish/Transformation.html

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Chapter 3 recombinant dna technology

  • 1. Recombinant DNA Technology BTEC3301
  • 2. Recombinant DNA Technology  Recombinant DNA technology procedures by which DNA from different species can be isolated, cut and spliced together -- new "recombinant " molecules are then multiplied in quantity in populations of rapidly dividing cells (e.g. bacteria, yeast).
  • 3. Recombinant DNA Technology  The term gene cloning, recombinant DNA technology and genetic engineering may seems similar, however they are different techniques in Biotechnology and they are interrelated
  • 4. Recombinant DNA Technology  Human gene therapy, genetically- engineered crop plants and transgenic mice have become possible because of the powerful techniques developed to manipulate nucleic acids and proteins.
  • 5. Recombinant DNA Technology  In the early 1970s it became possible to isolate a specific piece of DNA out of the millions of base pairs in a typical genome.
  • 6. Recombinant DNA Technology  Currently it is relatively easy to cut out a specific piece of DNA, produce a large number of copies , determine its nucleotide sequence, slightly alter it and then as a final step transfer it back into cell in.
  • 7. Recombinant DNA Technology Recombinant DNA technology is based on a number of important things:  Bacteria contain extrachromosomal molecules of DNA called plasmids which are circular.
  • 8. Recombinant DNA Technology  Bacteria also produce enzymes called restriction endonucleases that cut DNA molecules at specific places into many smaller fragments called restriction fragments.
  • 9. Recombinant DNA Technology Restriction Enzymes and plasmid  There are many different kinds of restriction endonucleases  Each nuclei cuts DNA at a specific site defined by a sequence of bases in the DNA called a recognition site
  • 10. Recombinant DNA Technology Restriction Enzymes and plasmid  A restriction enzyme cuts only double- helical segments that contain a particular sequence, and it makes its incisions only within that sequence--known as a "recognition sequence".
  • 11. Recombinant DNA Technology Restriction Enzymes and plasmid  Sticky end and blunt end are the two possible configurations resulting from the breaking of double-stranded DNA
  • 12. Recombinant DNA Technology Restriction Enzymes and plasmid  If two complementary strands of DNA are of equal length, then they will terminate in a blunt end, as in the following example:  5'-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT-3'  3'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA-5'
  • 13. Recombinant DNA Technology Restriction Enzymes and plasmid  However, if one strand extends beyond the complementary region, then the DNA is said to possess an overhang:  5'-ApTpCpTpGpApCpT-3'  3'-TpApGpApCpTpGpApCpTpApCpG-5'
  • 14. Recombinant DNA Technology Restriction Enzymes and plasmid  If another DNA fragment exists with a complementary overhang, then these two overhangs will tend to associate with each other and each strand is said to possess a sticky end:
  • 15. Recombinant DNA Technology Restriction Enzymes and plasmid  5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'  3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5' Becomes  5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'  3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'
  • 16. Recombinant DNA Technology Restriction Enzymes and plasmid  Restriction Enzymes are primarily found in bacteria and are given abbreviations based on genus and species of the bacteria.  One of the first restriction enzymes to be isolated was from EcoRI  EcoRI is so named because it was isolated from Escherichia coli strain called RY13.
  • 17. Recombinant DNA Technology Digestion of DNA by EcoRI to produce cohesive ends ( Fig. 3.1):
  • 18. Recombinant DNA Technology Creating recombinant DNA :  The first Recombinant DNA molecules were made by Paul Berg at Stanford University in 1972.  In 1973 Herbert Boyer and Stanley Cohen created the first recombinant DNA organisms.
  • 19. Recombinant DNA Technology Creating Recombinant DNA (Fig 3.2):
  • 20. Recombinant DNA Technology Reading materials :Summary of Recombinant DNA technology process:  Recombinant DNA technology requires DNA extraction, purification, and fragmentation.  Fragmentation of DNA is done by specific 'restriction' enzymes and is followed by sorting and isolation of fragments containing a particular gene.
  • 21. Recombinant DNA Technology Summary of Recombinant DNA technology process:  This portion of the DNA is then coupled to a carrier molecule.  The hybrid DNA is introduced into a chosen cell for reproduction and synthesis.
  • 22. Recombinant DNA Technology Transformation and Antibiotic Selection  Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign DNA.
  • 23. Recombinant DNA Technology Transformation and Antibiotic Selection  There are more aggressive techniques for inserting foreign DNA into eukaryotic cells. For example, through electroporation .  Electroporation involves applying a brief (milliseconds) pulse high voltage electricity to create tiny holes in the bacterial cell wall that allows DNA to enter.
  • 24. Recombinant DNA Technology Plasmids and Antibiotic resistance  Plasmids were discovered in the late sixties, and it was quickly realized that they could be used to amplify a gene of interest.  A plasmid containing resistance to an antibiotic (usually ampicillin) or Tetracycline, is used as a vector.
  • 25. Recombinant DNA Technology  The gene of interest (resistant to Ampicillin) is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that is sensitive to ampicillin.( Text bk:Pg 58)  The bacteria are then spread over a plate that contains ampicillin.
  • 26. Recombinant DNA Technology Plasmids and Antibiotic resistance  The ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate.  Those bacteria which do not acquire the plasmid with the inserted gene of interest will die.
  • 27. Recombinant DNA Technology Plasmids and Antibiotic resistance  As long as the bacteria grow in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with the gene of interest that has been inserted to the plasmid .
  • 28. Recombinant DNA Technology Fig 3.3 (a). Selecting a Gene in a plasmid and Antibiotic selection.
  • 29. Recombinant DNA Technology Assignment: For above procedure, Read Transformation of Bacterial cells and Antibiotic selection pg 61.
  • 30. Recombinant DNA Technology Human Gene cloning  Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen replicas.
  • 32. Recombinant DNA Technology Reading materials: Summary of Recombinant DNA and Cloning (Fig. below):  Isolation of two kinds of DNA  Treatment of plasmid and foreign DNA with the same restriction enzyme  Mixture of foreign DNA with plasmids
  • 33. Recombinant DNA Technology  Addition of DNA ligase  Introduction of recombinant plasmid into bacterial cells  Production of multiple gene copies by gene cloning
  • 34. Recombinant DNA Technology Summary of Recombinant DNA and Cloning (Fig.):
  • 35. Recombinant DNA Technology  This segment is "glued" into place using an enzyme called DNA ligase.  The result is an edited, or recombinant, DNA molecule.
  • 36. Recombinant DNA Technology  When this recombinant plasmid DNA is inserted into E. coli, the cell will be able to process the instructions to assemble the amino acids for insulin production.
  • 37. Recombinant DNA Technology  More importantly, the new instructions are passed along to the next generation of E. coli cells in the process known as gene cloning.  Assignment: Human gene cloning pg 63
  • 38. Recombinant DNA Technology Fig: Inserting a DNA sample into a Plasmid
  • 39. Recombinant DNA Technology References  http://en.wikipedia.org/wiki/Restriction_enzyme  http://web.mit.edu/esgbio/www/rdna/cloning.html  http://faculty.plattsburgh.edu/donald.slish/Transformation.html