Download - Recombinant dna-1
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Hoàng Thị Mỹ Hạnh
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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).
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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…
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Recombinant DNA Tool
Vector
Enzyme used in molecular biology
Host cells
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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.
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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
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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.
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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”.
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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
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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.
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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’
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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’
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Compatible sticky ends
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Restriction Endonucleases Cleave DNA at specific DNA sequences
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Converting sticky ends to blunt ends
Filling in Trimming back
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Converting blunt end to sticky end by using linker
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Converting blunt end to sticky end by using linker
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DNA Ligase in Action!
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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.
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Gel: Agarose or Acrylamide
Dye
Nucleic acid stain: EtBr
Marker
Running time
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Electrophoretic mobility of form of plasmid DNA
Circular DNA
Linear DNA
Supercoil DNA
Hypersupercoil DNA
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Uncut plasmid DNA can be in five forms
Nicked
Circular
Linear covalently close
Supercoil
Hyper supercoil
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