Restriction Digestion and Analysis of Lambda DNA Kit

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Restriction Digestion and Analysis of Lambda DNA
Kit
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What can you do with the Restriction Digestion
and Analysis of Lambda DNA Kit?

• Understand the use of restriction enzymes as
  biotechnology tools
• Become familiar with principals and techniques of
  agarose gel electrophoresis
• Generate a standard curve from a series of DNA
  size fragments
• Estimate DNA fragment sizes from agarose gel data

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What are restriction enzymes?

• Evolved by bacteria to protect against viral DNA
  infection
• Endonucleases cleave within DNA strands
• 3,139 known enzymes

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How does it work?

• Enzyme Site Recognition
• Each enzyme digests (cuts) DNA at a specific
  sequence ? restriction site
• Enzymes recognize 4-, 6- or 8- base pair,
  palindromic sequences
• Isoschizomers recognize identical sequences, but
  have different optimum reaction conditions and
  stabilities
• Can be unambiguous or ambiguous

Unambiguous
Ambiguous
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Palindromic Sequences

• 5 versus 3 overhang Sticky Ends

Enzyme cuts ?
Generates 5 overhang

• 5 and 3 versus Blunt ends

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Common Restriction Enzymes

• EcoRI
• Escherichia coli
• 5 overhang
• HindIII
• Haemophilus influensae
• 5 overhang
• PstI
• Providencia stuartii
• 3 overhang

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What is needed for restriction digestion?

• Template DNA, uncut DNA, often bacterial phage
  DNA
• DNA standard or marker, a restriction enzyme of
  known fragment sizes
• Restriction enzyme(s), to cut template DNA
• Restriction Buffer, to provide optimal conditions
  for digestion

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Lambda Phage DNA

• Genomic DNA of a bacterial virus
• Attacks bacteria by inserting its nucleic acid
  into the host bacterial cell
• Replicates rapidly inside host cells until the
  cells burst and release more phages
• Harmless to man and other eukaryotic organisms

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Restriction Enzyme Digestion

• Restriction Buffer provides optimal conditions
• NaCl provides the correct ionic strength
• Tris-HCl provides the proper pH
• Mg2 is an enzyme co-factor

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DNA Digestion Temperature

• Why incubate at 37?C?
• Body temperature is optimal for these and most
  other enzymes
• What happens if temperature is too hot or cool?
• Too hot enzyme may be denatured, killed
• Too cool enzyme activity lowered, requiring
  longer digestion time

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Agarose Gel Electrophoresis

• Electrolysis the splitting of water using
  electricity
• current splits water into hydrogen ions (H) and
  hydroxyl ions (OH-)
• Electrophoresis a method of separating charged
  molecules in an electrical field DNA has an
  overall negative charge
• Used to separate DNA fragments by size

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Components of an Electrophoresis System

• Power supply and chamber, a source of negatively
  charged particles with a cathode and anode
• Buffer, a fluid mixture of water and ions
• Agarose gel, a porous material that DNA migrates
  through
• Gel casting materials
• DNA ladder, mixture of DNA fragments of known
  lengths
• Loading dye, contains a dense material and allows
  visualization of DNA migration
• DNA Stain, allows visualizations of DNA fragments
  after electrophoresis

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Bio-Rads Electrophoresis Equipment
Precast Ready Agarose Gel
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Electrophoresis Buffer

• TAE (Tris-acetate-EDTA) and TBE
  (Tris-borate-EDTA) are the most common buffers
  for duplex DNA
• Establish pH and provide ions to support
  conductivity
• Concentration affects DNA migration
• Use of water will produce no migraton
• High buffer conc. could melt the agarose gel

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Agarose Gel

• A porous material derived from red seaweed
• Acts as a sieve for separating DNA fragments
  smaller fragments travel faster than large
  fragments
• Concentration affects DNA migration
• Low conc. larger pores? better resolution of
  larger DNA fragments
• High conc. smaller pores? better resolution of
  smaller DNA fragments

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DNA Staining

• Allows DNA visualization after gel
  electrophoresis
• Ethidium Bromide
• Bio-Safe DNA stains

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Complete a Gel Electrophoresis simulation
at http//gslc.genetics.utah.edu/units/biotech/g
el/
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Restriction Enzyme Digest and Analysis Procedures
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Actual Results of Restriction Enzyme Digestion

• Lane 1, DNA markers (HindIII lambda digest)
• lane 2, uncut lambda DNA
• lane 3, lambda DNA digested with PstI
• lane 4, lambda DNA digested with EcoRI
• lane 5, lambda DNA digested with HindIII

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Analysis of DNA Fragments

• Determine restriction fragment sizes
• Create standard curve using DNA marker
• Measure distance traveled by restriction
  fragments
• Determine size of DNA fragments

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DNA Marker Standard Curve

• Size (bp) Distance (mm)
• 23,000 11.0
• 9,400 13.0
• 6,500 15.0
• 4,400 18.0
• 2,300 23.0
• 2,000 24.0

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Factors Affecting Restriction Enzyme Digestion

• Temperature, restriction enzymes are sensitive to
  prolonged periods of exposure to heat
• Cross contamination of restriction enzymes
• Buffer, optimum pH
• Incubation temperature, maintain optimum
  temperature during restriction enzyme activity
• And FinallyDont forget to ADD your restriction
  enzyme to the reaction!!!