Recombinant DNA Technology

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Recombinant DNA Technology

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rDNA Technology

• Restriction Enzymes and DNA Ligase
• Plasmid Cloning Vectors
• Transformation of Bacteria
• Blotting Techniques

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Restriction Enzymes

• Most significant advancement permitting rDNA
  manipulation
• Differ from other nucleases
• recognize and cleave a specific DNA sequence
  (Type II restriction enzymes)

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Restriction Enzymes

• Nomenclature
• EcoRI
• E Escherichia genus name
• co coli species name
• R strain RY12 strain or serotype
• I Roman numeral one first enzyme
• HinDIII
• Haemophilus influenza serotype d 3rd
  enzyme

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Restriction Enzymes

• Recognition sites
• Generally 4, 6, or 8 bp in length
• Most sites are palindromic
• OTTO / HANNAH / REGAL LAGER
• A MAN A PLAN A CANAL PANAMA
• For REases - sequence reads the same in a
  5---gt3 direction on each strand

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Restriction Enzymes

• EcoRI
• 5 GAATTC 3
• 3 CTTAAG 5
• Hind III
• 5 AAGCTT 3
• 3 TTCGAA 5

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Restriction Enzymes

• Cleave DNA to generate different ends
• Staggered cut
• 5 extension
• 3 extension
• Blunt end

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Staggered Cut / 5 or 3 Extension
Eco RI
5G AATTC CTTAA
G
5GAATTC CTTAAG

Pst I
5 CTGCA G G
ACGTC
5 CTGCAG GACGTC

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Restriction Enzymes in DNA Cloning

• How are REases used ?
• Ends are sticky
• Complementary
• Any two DNAs cut with same enzyme can stick
  together through complementary base pairing

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Annealing sticky ends
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Annealing Sticky ends
DNA strands held together only by
basepairing Nicks in strands need to be repaired
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Linking Restriction Fragments

• T4 DNA Ligase
• repairs nicks in DNA strands
• (reforms phosphodiester bond)
• uses energy from ATP
• works on blunt or sticky ends
• Other enzymes used in rDNA technology

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T4 DNA Ligase Mode of Action
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rDNA Technology

• Restriction Enzymes and DNA Ligase
• Plasmid Cloning Vectors
• Transformation of Bacteria
• Creating and Screening Genomic Libraries
• cDNA Library Construction
• Vectors for Cloning Large Pieces of DNA
• Blotting Techniques

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Recombinant DNA Cloning Procedure
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Recombinant DNA Cloning Procedure

1) Enzymatic digestion
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Recombinant DNA Cloning Procedure

2) Ligation of Target and vector
DNA Ligase
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Recombinant DNA Cloning Procedure
3) Transform Ligated DNA into Bacteria
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Plasmid Cloning Vectors

• Recombinant DNA needs to be replicated in
  bacterial cell
• Self-replicating piece of DNA
• termed cloning vehicle
• can be plasmid or phage

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Plasmid Cloning Vectors

• Small circular piece of DNA
• Exists separate from chromosome
• Derived from naturally occurring plasmids
• High copy number 10-100 copies / cell
• Low copy number 1-4 copies / cell

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Plasmid Cloning Vectors

• Derived from naturally occurring plasmids
• Altered features
• small size (removal of non-essential DNA)
• higher transformation efficiency
• unique restriction enzyme sites
• one or more selectable markers
• origin of replication (retained from original
  plasmid)
• other features promoters, etc.

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pBR322old-style general purpose plasmid
4362 bp
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pUC19
2.68kbp
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Multiple Cloning Sequence (Polylinker)
EcoRI
KpnI
BamHI
Sal I
GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGAC
XbaI
SmaI
SacI
Part of MCS of pUC19 and other plasmids
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Plasmid Cloning Vehicles
Some plasmids (Expression Plasmids) have
promoters upstream of cloning sites for
expression of genetic info encoded by DNA
fragment
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Shuttle Plasmid Cloning Vehicles
Some plasmids (Shuttle Plasmids) have origins of
replication for E. coli and another organism
yeast, mammalian cells or other bacteria
E. coli ori
yeast ori mammalian ori
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Plasmid Cloning Vehicles

• What prevents plasmid DNA from reforming during
  ligation?

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Plasmid Cloning Vehicles

• What prevents plasmid DNA from reforming during
  ligation and transforming cells as do the
  recombinant molecules?
• Three ways to prevent
• Treat with Alkaline Phosphatase
• Directional Cloning
• Suicide Plasmids with ccdB gene

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Plasmid Cloning Vehicles

• Alkaline Phosphatase
• removes 5 PO4 from end of DNA strand
• prevents formation of new phosphodiester bond by
  DNA Ligase

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Alkaline Phosphatase Action
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Alkaline Phosphatase Action
Two nicks remain Will be repaired in bacterial
cell follow- ing transformation
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Directional Cloning

• Digest plasmid and target DNA with two different
  restriction enzymes
• Hind III and BamHI
• Ends are not compatible (cant basepair)
• Plasmid wont re-circularize unless target DNA
  has inserted

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Transformation of Bacteria

• rDNA constructed in the lab must be introduced
  into host cell
• Cells must be able to take up DNA - COMPETENT
• Growing bacteria will produce lots of copies of
  the DNA

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Transformation of Bacteria

• Two basic methods to produce competent bacteria
  (able to take up added DNA)
• Chemical competent
• Electroporation

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Transformation of Bacteria

• Chemical competent
• Divalent metal ion Ca , required
• treat cells with ice-cold CaCl2 solutions
• Ca ions alter membrane so it is permeable to
  DNA

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Transformation of Bacteria

• Electroporation
• Cell/DNA mix given high voltage electric shock
• 2.5kvolts, 5msec
• useful for high efficiency transformation
• 109 transformants / µg of DNA

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Transformation of Bacteria

• Both methods are very inefficient
• only a few of cells actually take up DNA
• How are the transformed cells selected?
• antibiotic resistance gene on plasmid
• ampicilin, tetracycline, chloramphenicol, etc.
• transformed cells grow non-transformed die

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Immunological Screen of Library
detectable product
substrate
reporter enzyme
2 Ab
1 Ab
target protein
matrix
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Immunological Screen
master plate
protein
matrix
cells
1
2
transfer cells
lyse cells bind protein
subculture cells from master plate
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Add 1Ab wash
positive signal
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5
Add substrate
Add 2Ab-Enzyme wash
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Blotting Techniques

• Several techniques for size fraction of nucleic
  acid fragments and proteins
• Nucleic Acids
• Agarose Gels
• Polyacrylamide Gels - higher resolution
• Proteins
• SDS PAGE - denatured proteins

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Blotting Techniques

• How can one fragment be detected in a complex
  mixture?
• Transfer the macromolecule to a membrane
• Detect with
• complementary nucleic acid probe or
• with an antibody to the specific protein

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Blotting Techniques
Blot Type Matrix Molecule
Detection Southern agarose DNA nucleic
acid northern agarose RNA nucleic
acid western polyacrylamide Protein
antibody
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Blotting Techniques Info Obtained

• Southern Blots
• presence of fragment (gene)
• of fragments (approx. of genes)
• sizes of fragments
• sequence similarity between target probe

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Blotting Techniques Info Obtained

• Northern blots
• presence of RNA in tissue
• level of expression
• size of mRNA
• sequence similarity between target probe

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Blotting Techniques Info Obtained

• Western blots
• presence of protein in tissue
• level of expression
• size of protein