GEN 314

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GEN 314

• Gene Manipulation Lecture One

Frank M. Maleka Office 164 Biology Building
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Prescribed text OLD, R. W. and S. B. PRIMROSE
(1995). Principles of Gene Manipulation an
introduction to genetic engineering, 5th ed.
Suggested reading to be noted

• References

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Chapters to be covered!

• Chapters 1 2 Introduction and Basic
  techniques
• Chapter 3 Cutting and joining DNA molecules
• Chapter 4 Plasmids as cloning vehicles
• Chapter 6 Cloning strategies, gene libraries
  and cDNA cloning
• Chapter 7 Recombinant selection and screening
• Chapter 8 Expression in E. coli of cloned DNA
  molecules
• Chapter 10 Polymerase Chain Reaction (PCR)
• Chapter 14 Gene transfer to plants
• Chapter 17 The impact of recombinant DNA
  technology

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Introduction

• What is gene manipulation?
• Isolating a specific stretch of DNA sequence from
  a host organism
• Modification of the isolated DNA sequence via
  genetic or other methods
• Inserting the modified DNA sequence into the same
  or new host organism
• However, in some cases, isolated DNA sequence is
  not modified
• depends on the objective of the experiment
• Commonly involve technique termed gene cloning

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Basic tools

• DNA sequence! (RNA/protein)
• Restriction enzymes cut and paste tools
• Plasmids cloning vehicles
• Technique/s for selecting and screening
  recombinants
• gel electrophoresis
• blotting techniques

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Gel electrophoresis

• Process employed to resolve DNA fragments based
  on size separation
• commonly use agarose (or polyacrylamide),
  polysaccharide like agar or pectin that dissolves
  in boiling water and gels as it cools
• Application
• w/v concentration (e.g. 1g/100ml of buffer)
• add DNA (stained with Ethidium Bromide, EtBr)
  to a slab of gelled agarose
• apply an electric current across the gel
• DNA is negatively charged, migration towards
  positive electrode
• Rate of migration
• fragment size dependent small fragments
  migrate faster than larger fragments
• affected by shape of DNA circular fragments
  migrate differently from linear fragments of
  similar molecular weight (see later)
• agarose gel concentration the higher the
  concentration, the more it retards DNA movement
  through the gel
• voltage

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Gel electrophoresis
Klug et al. (2006), Fig. 19-22, pg. 472
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Nucleic acid blotting

• Blotting immobilization of sample nucleic acids
  onto a solid support, typically nylon filters or
  nitrocellulose membranes
• Three types of blots
• Southern blot (Edward Southern 1975, 1979)
  used to characterize the number, size,
  organization and sequence content of DNA sequence
  in the genome
• DNA-ssDNA hybridization

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Blotting
Klug et al. (2006), Fig. 19-24, pg. 475
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Other blots

• Northern blot (Alwine et al. 1979)
• used to provide information on the expression
  of specific genes and patterns of expression
  among different cellular tissues
• mRNA-cDNA hybridization
• Western blot (Burnette 1981)
• used to provide information on protein
  expression
• Protein-antibody hybridization
• Polyacrylamide gel electrophoresis

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Cutting DNA molecules

• Cutting a duplex DNA molecule restriction
  endonucleases
• Enzymes that recognize specific base sequences in
  a DNA molecule and cut the DNA at or near the
  recognized DNA sequence
• Discovery based on host-controlled restriction
  and modification systems in bacteria
• Host-controlled restriction system (e.g. phage ?
  and E. coli strains C and K) monitoring
  exogenous DNA, destroy if recognized as foreign
  (restriction endonuclease activity)
• Modification methylation of specific bases
  (recognition sequence) in the endogenous DNA to
  protect against restriction endonucleases

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Restriction endonucleases
See Table 3.1, pg 27 Roberts et al. (2004) Nuc.
Acids Res. 31 (7) 1805-1812

• Type I random cleavage sites
• require Mg2 and co-factors ATP and SAM
• single enzyme with three subunits, i.e.
  specificity, modification and restriction
  subunits
• cleavage occurs only if both strands are
  unmethylated, up to several kilobases away from
  the recognition site (not suitable for gene
  manipulation)
• Type II (most common type) cleave symmetric
  sequences
• single homodimer, require only Mg2 and no
  co-factors
• recognize a target sequence and cleave DNA
  within or near the target sequence
• produce discrete DNA fragments of defined
  length and sequence (suitable for gene
  manipulation)
• Type III (relatively rare type) cleave
  asymmetric sequences
• require Mg2 and co-factors ATP and SAM
• complex of two subunits, i.e. M subunit (site
  recognition and modification) and R subunit
  (nuclease activity)
• cleavage occurs a few bases (ca. 24bp 26bp)
  downstream of the recognition sequence

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Nomenclature

• Naming first letter of genus name and first two
  letters of species name (italics) e.g.
  Haemophilus influenzae Hin Escherichia coli
  Eco
• Strain ID in subscript e.g. Hind Ecok
• Particular host strain having several restriction
  and modification systems, use roman numerals (?
  res. endonuc. types) e.g. Hind I, Hind II, Hind
  III, etc
• In practice, abbreviations written on the same
  line e.g. HindIII
• Again, Roberts et al. (2004) Nuc. Acids Res. 31
  (7) 1805-1812

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Target sites

• Sticky or cohesive ends
• fragments with protruding 5-termini, e.g.
  BamHI
• alternatively, protruding 3-termini such as
  PstI
• Others produce blunt ends, e.g. SmaI
• Different enzymes recognize DNA sequences of
  different lengths, i.e. 4-bp, 5-bp, 6-bp, 7bp,
  etc.
• Implications for application, 44 256 bp
  (frequent cutters) or 46 4096 bp (rare cutters)

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Joining DNA molecules

• DNA ligase seals single-stranded nicks between
  adjacent nucleotides (formation of phosphodiester
  bonds)
• DNA ligase from E. coli and phage T4 are very
  similar, except in their co-factor requirements
  NAD and ATP, respectively (see Fig 3.4, pg 37)

Klug et al. (2006), Fig. 19-4, pg. 460
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Related methods

• Double-linkers
• Adaptors
• Homopolymer tailing uses terminal
  deoxynucleotidyltransferase to synthesize
  homopolymeric 3-single-stranded tails at the
  ends of fragments (blunt to cohesive)
• adds nucleotides to the 3-OH terminus of DNA
  molecule
• application in cDNA synthesis (see later)

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Plasmids as cloning vehicles

• Replicons that are inherited in an
  extrachromosomal state
• ability to replicate independently of
  chromosomes
• Most plasmids exist as double-stranded circular
  DNA molecules
• intact circles covalently closed circles
  (CCC DNA)
• only one strand intact open circles (OC DNA)
• CCC DNA often form supercoiled configuration
  due to deficiency of turns in the double helix
  (also affected by the EtBr)
• different structural config. allow separation
  during electrophoresis (Fig. 4.2, pg 48)
• Two major types of plasmids (tra genes that
  promote bacterial conjugation)
• conjugative high molecular weight and found
  as one to three copies per chr (stringent
  plasmids)
• non-conjugative low molecular wt and found
  as multiple copies/chr (relaxed plasmids)

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Prerequisites for plasmids as cloning vehicles

• Purification of plasmid DNA
• CsCl (and EtBr) purification method
• alkaline extraction method (Birnboim and Doly
  1979)
• Low molecular weight
• easy to handle
• multiple copies
• minimizes the chances of the vector to have
  multiple substrate sites for any r.e.
• Ability to confer readily selectable phenotypic
  traits on host cells (Table 4.1 pg 49)
• Single sites for a number of r.e.s, preferably
  in genes conferring readily scorable phenotype
• insertional inactivation

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End Lecture One