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Hoàng Thị Mỹ Hạnh
Recombinant DNA Technology
 Recombinant DNA technology procedures by which
DNA from different species can be isolated, cu...
Application of Recombinant DNA
 Human gene therapy: recombinant human insulin,
recombinant human growth factor, recombina...
Recombinant DNA Tool
Vector
Enzyme used in molecular biology
Host cells
Restriction enzyme Recognize and cut at DNA specific sequence
DNA ligase Join compatible ends of DNA fragment. Use
ATP
Alk...
 Restriction enzymes are primarily found in bacteria
and are given abbreviations based on genus and
species of the bacter...
Classify restriction enzymes
 Type I enzymes are complex, multisubunit, combination restriction-and-
modification enzymes...
Restriction Enzymes
 Bacteria have learned to "restrict" the possibility of
attack from foreign DNA by means of "restrict...
Type II restriction enzyme
 EcoRI – Escherichia coli strain R, 1st enzyme
 BamHI – Bacillus amyloliquefaciens strain H, ...
Basics of type II Restriction Enzymes
 No ATP requirement.
 Recognition sites in double stranded DNA have a 2-fold
axis ...
Results of Type II Digestion
 Enzymes with staggered cuts  complementary ends
 HindIII - leaves 5´ overhangs (“sticky”)...
Results of Type II Digestion
 Enzymes that cut at same position on both strands
leave “blunt” ends
 SmaI
 Isochizomer X...
Compatible sticky ends
Restriction Endonucleases Cleave DNA
at specific DNA sequences
Converting sticky ends to blunt ends
Filling in
Trimming back
Converting blunt end to sticky end by using
linker
Converting blunt end to sticky end by using
linker
DNA Ligase in Action!
Electrophoresis
 Gel electrophoresis is a method for separation and
analysis of macromolecules (DNA, RNA and proteins)
an...
 Gel: Agarose or Acrylamide
 Dye
 Nucleic acid stain: EtBr
 Marker
 Running time
Electrophoretic mobility of form of
plasmid DNA
 Circular DNA
 Linear DNA
 Supercoil DNA
 Hypersupercoil DNA
Uncut plasmid DNA can be in five forms
 Nicked
 Circular
 Linear covalently close
 Supercoil
 Hyper supercoil
Recombinant dna-1
Recombinant dna-1
Recombinant dna-1
Recombinant dna-1
Recombinant dna-1
Recombinant dna-1
Recombinant dna-1
Recombinant dna-1
Recombinant dna-1
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Recombinant dna-1

Written by Dr Hoang Thi My Hanh
VNU University of Science

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Recombinant dna-1

  1. 1. Hoàng Thị Mỹ Hạnh
  2. 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. 3. Application of Recombinant DNA  Human gene therapy: recombinant human insulin, recombinant human growth factor, recombinant hepatitis B vaccine…  Engineered crop plants: golden rice (β-carotene), herbicide resistant crops, insect resistant crops…
  4. 4. Recombinant DNA Tool Vector Enzyme used in molecular biology Host cells
  5. 5. Restriction enzyme Recognize and cut at DNA specific sequence DNA ligase Join compatible ends of DNA fragment. Use ATP Alkaline phosphatase Remove phosphate group from strand of DNA Polynucleotide kinase Add phosphate group to a DNA strand in the 5’ to 3’ direction DNA polymeraseI DNA synthesize Exonuclease III Digest nucleotide from a DNA strand in the 3’ to 5’ direction RNAse Nuclease digest RNA, not DNA Taq DNA polymerase Heat-stable DNA polymerase isolated from thermostable microbe.
  6. 6.  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. Restriction Enzymes
  7. 7. Classify restriction enzymes  Type I enzymes are complex, multisubunit, combination restriction-and- modification enzymes that cut DNA at random far from their recognition sequences.  Type II enzymes cut DNA at defined positions close to or within their recognition sequences.  Type III enzymes are also large combination restriction-and-modification enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests.  Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr systems of E. coli.
  8. 8. Restriction Enzymes  Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes”.  Cut up “foreign” DNA that invades the cell.  Type II and III restriction enzymes cleave DNA chains at selected sites.  Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage.  An enzyme that recognizes a 6-base sequence is called a "six-base cutter”.
  9. 9. Type II restriction enzyme  EcoRI – Escherichia coli strain R, 1st enzyme  BamHI – Bacillus amyloliquefaciens strain H, 1st enzyme  DpnI – Diplococcus pneumoniae, 1st enzyme  HindIII – Haemophilus influenzae, strain D, 3rd enzyme  BglII – Bacillus globigii, 2nd enzyme  PstI – Providencia stuartii 164, 1st enzyme  Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme  KpnI – Klebsiella pneumoniae, 1st enzyme
  10. 10. Basics of type II Restriction Enzymes  No ATP requirement.  Recognition sites in double stranded DNA have a 2-fold axis of symmetry – a “palindrome”.  Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends.
  11. 11. Results of Type II Digestion  Enzymes with staggered cuts  complementary ends  HindIII - leaves 5´ overhangs (“sticky”) 5’ --AAGCTT-- 3’ 5’ --A AGCTT--3’ 3’ --TTCGAA-- 5’ 3’ –TTCGA A--5’  KpnI leaves 3´ overhangs (“sticky”) 5’--GGTACC-- 3’ 5’ –GGTAC C-- 3’ 3’--CCATGG-- 5’ 3’ –C CATGG-- 5’
  12. 12. Results of Type II Digestion  Enzymes that cut at same position on both strands leave “blunt” ends  SmaI  Isochizomer XmarI 5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’ 3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’
  13. 13. Compatible sticky ends
  14. 14. Restriction Endonucleases Cleave DNA at specific DNA sequences
  15. 15. Converting sticky ends to blunt ends Filling in Trimming back
  16. 16. Converting blunt end to sticky end by using linker
  17. 17. Converting blunt end to sticky end by using linker
  18. 18. DNA Ligase in Action!
  19. 19. Electrophoresis  Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.
  20. 20.  Gel: Agarose or Acrylamide  Dye  Nucleic acid stain: EtBr  Marker  Running time
  21. 21. Electrophoretic mobility of form of plasmid DNA  Circular DNA  Linear DNA  Supercoil DNA  Hypersupercoil DNA
  22. 22. Uncut plasmid DNA can be in five forms  Nicked  Circular  Linear covalently close  Supercoil  Hyper supercoil

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