Gene Cloning

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1. Fragment of DNA to be cloned is inserted into
   vector to produce rDNA
2. Vector to transport gene into host cell e.g.
   plasmid or bacteriophage chromosome
3. Multiplication of rDNA molecule together with
   vector
4. Division of host cell
5. After a large number of cell divisions forms a
   colony or clone with identical genes

Gene Cloning
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PCR
Thermal Cycler

1. Heat to 94 oC to denature the double strand DNA
2. Cool to 55 oC for primers to anneal to DNA at
   specific position (gene of interest)
3. Raise to 74 oC for Taq DNA polymerase to function
4. Synthesis of DNA strands complementary to
   template DNA molecules in opposite direction
5. Repeat the cycle 25-30 times

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Importance of Gene Cloning and PCR

• Useful in gene isolation
• E.Coli contains over 4000 different genes!
• PCR in a few hours, gene cloning in weeks!
• Limitations of PCR
• Sequence of annealing sites must be known in
  order for primers to anneal to correct positions
• Limit to length of DNA sequence i.e. 5 kilobases
  40 kilobases

Application of PCR Useful to detect or isolate
gene sequences already known eg. Test for
mutation in blood using globin genes or use of
primers specific for the DNA of a harmful virus
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Plasmids

• Circular molecules of DNA that lead independent
  existence in host
• Carry genes that are responsible for a useful
  characteristic displayed by host bacterium
• Survival in normally toxic concentrations of
  antibiotics antibiotic resistance as a
  selectable marker
• Example ampicillin, tetracycline, kanamycin
  resistant gene RP4
• At least one DNA sequence that can act as an
  origin of replication
• OR
• Integrate into bacterial chromosome for division
• lt 10kb desirable for a cloning vehicle

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Plasmid Classification

1. Fertility or F plasmids carry only tra genes
   and have no characteristic beyond the ability to
   promote conjugal plasmid transfer
2. Resistance or R plasmids confers resistance to
   one or more antibacterial agents
3. Col plasmids code for colicins, proteins that
   kill other bacteria
4. Degradative plasmids allow the host bacterium to
   metabolize unusual molecules e.g. toluene and Hg
5. Virulence plasmids confer pathogenicity on the
   host bacterium e.g. Ti plasmids of agrobacterium
   tumefaciens induce crown gall disease on plants

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total cell DNA pure plasmid DNA
Purification of DNA from living cells

• 3.1 Preparation of total cell DNA
• A culture of bacteria is grown and then harvested
• The cells are removed and broken to give a cell
  extract
• The DNA is purified from the cell extract
• The DNA is concentrated

• 3.1.1 Growing and harvesting a bacterial culture
• culture bacteria in a liquid both
• 2 types of growth media defined medium and
  undefined medium
• Define media is used when the bacteria culture
  has to be grown under precisely controlled
  conditions e.g. M9
• Undefined media is used when culture is grown
  for a source of DNA e.g. Luria-Bertani (LB)
  contain yeast and tryptone

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• 3.1.2 Preparation of a cell extract
• Purpose is to break open bacterial cells (cell
  lysis) by either physical or chemical means
• lysozyme digest polymeric compunds that define a
  cell walls rigidity
• EDTA remove Mg2 that is essential for the
  structure of cell envelope
• Detergents e.g. SDS help to remove lipid
  molecules and cause disruption of cell membranes
• Centrifugation to remove cell debris that settle
  at the bottom

• 3.1.3 Purification of DNA from a cell extract
• Bacterial extract can contain a lot of protein
  and RNA inaddtion to desired DNA
• Add phenol or 11 mixture of phenol and
  chloroform. Ppt proteins leaving behind DNA RNA
  and can be seperated after centrifugation
• Cell extracts that contain a large amount of
  proteins must be treated with Protease such as
  proteinase K before addition of phenol
• Degrade RNA with suitable ribonuclease

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• 3.1.4 Measurement of DNA concentration
• UV absorbance spectrophotometry
• Absorbance at 260 nm (A260) of 1.0 50ug of
  double strand DNA/ml
• UV absorbance can also check for purity of DNA
  preparation A260/A280 1.8 for pure samples

• 3.1.5 Preparation of total cell DNA from
  non-bacteria organism
• plant tissues contain large amount of
  carbohydrates that cannot be removed by phenol
  extraction
• METHOD 1
• Add CTAB (cetyltrimethylammonium bromide) so that
  CTAB complex ppt out together with the nucleic
  acid. Carbohydrates and proteins and other
  contaminants as a supernatant. Centrifuge and
  collect the ppt.
• Nucleic acids remaining can be concentrated
  using ethanol precipitation and RNA remove by
  ribonuclease treatment

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• METHOD 2
• Add guanidinium thiocyanate to dissolve all
  biochemical other than nucleic acids
• Pass the sample through a chromatography column
  with silica particles inside. DNA in presence of
  guanidinium thiocyanate bind more strongly to
  silica
• DNA is recovered by adding water which
  destabilizes interaction between DNA and silica

• 3.2.1 Plasmid Separation
• Separation by size work on the principle that
  cells are lysed under very carefully controlled
  conditions
• very little breakage of DNA chromosome. Hence
  DNA is much larger than plasmid and can be
  removed together with cell debris in
  centrifugation
• Chromosomal DNA also attached to cell envelope
  and settle at bottom

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• Method 1
• For E. Coli and related species under controlled
  lysis
• Add EDTA and lysozyme in the presence of 25
  sucrose prevent cell from bursting immediately
• Cell lysis is induced by adding non-ionic
  detergent (Triton X-100) because ionic detergent
  cause chromosomal breakage
• Centrifugation leaves a cleared lysate consisting
  of only plasmid DNA

• Method 2
• Separation by conformation using alkaline
  denaturation.
• Plasmid is circular DNA but also often
  supercoiled
• A narrow pH range at which non-supercoiled DNA is
  denatured while supercoiled plasmid will not. pH
  range between 12.0 12.5 (using NaOH)
• After non supercoiled DNAs H bonding is broken
  to form linear strand DNA, add acid to reach pH
  7.0
• Denatured bacteria DNA strands entangle into a
  mass and can be centrifuged, leaving pure plasmid
  DNA in supernatant

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• 3.2.2 Plasmid Amplification
• To increase the copy number of plasmid
• some multicopy plasmid can replicate in the
  absence of protein synthesis, whereas main
  bacteria chromosome cannot replicate
• After a satisfactory cell density is reached,
  add inhibitor of protein synthesis e.g.
  chloramphenicol and incubate for another 12 hrs
• Plasmid copy of 1000 , hence an efficient way
  of increasing yield of multicopy plasmid

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Chapter 4 manipulation of purified DNA

• DNA manipulative enzymes
• Nuclease cut or degrade nucleic acid
• exonuclease remove nucleotides one at a time
  from end of DNA
• endonuclease break internal phosphodiester bonds
  within DNA
• Ligase join nucleic acid
• Polymerase make copies of molecules
• Modifying enzymes remove or add chemical grp
• alkaline phosphatase remove phosphate grp at 5
  end of DNA
• Polynucleotide kinase reverse effect to alkaline
  phosphatase
• Terminal deoxynucleotidyl transferase add
  deoxyribonucleotides to 3 end of DNA
• Topoisomerase introduce or remove supercoils
  from covalently closed circular DNA

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Chapter 5 Introduction of DNA into living cells

• 5.1.1 Transformation - Uptake of foreign DNA
  molecule by a cell
• Most cell take only limited amounts of DNA
  normally, must increase efficiency of intake by
  physical or chemical enhancement
• E.coli cells soaked in ice cold salt solution
  more efficient at DNA uptake. A solution of 50mM
  CaCl2 is used
• Next heat shock the solution to 42 oC for 2 min
  to facilitate uptake of plasmid by cell
• 5.1.2 Selection for transformed cells
• using selectable marker e.g. amipicillin
  resistance gene or tetracycline resistance gene
• LacZ gene codes for beta galactosidase, breaks
  lactose to glucose galactose
• e.g. PUC8 plasmid with both AmpR and LacZ genes
• some strains of E.coli have modified lacZ gene
  that lack the segment LacZ

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• These mutants can only synthesize beta
  galactosidase if it has PUC8 plasmid that carries
  the missing LacZ gene segment
• Add X-gal (5-bromo-4-chloro-3-indoyl-B-D-galactop
  yra-lactose) which is broken down by B
  galactosidase to form a blue product
• Add IPTG (inducer of the beta galactosidase)
• Xgal IPTG agar plate to select cells
  between white and blue colonies

Cloning vectors for E.Coli
pBR322 ori, ampR, tetR pBR327 ori, ampR,
tetR pUC8 ori, ampR, lacZ

• Nomenclature of plasmid cloning vectors
• pBR322
• p plasmid
• BR identifies the laboratory in which vector
  was discovered (BR for Bolivar and Rodriguez, the
  2 researchers that developed it)
• 322 distinguishes this plasmid from others
  developed in the same laboratory (pBR325, pBR327
  etc..)