PFGE Pulsed Field Gel Electrophoresis

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PFGE - Pulsed Field Gel Electrophoresis

• Horizontal gel electrophoresis is the most common
  means of separating DNA molecules 0.1 to 30 kb in
  size. This technique employs a continuous and
  homogeneous electrical field and an agarose
  matrix to achieve separation. Since all DNA
  molecules have a similar chargemass ratio and
  hence, a similar velocity when subjected to a
  voltage gradient in an aqueous solution, it is
  the sieving properties of agarose gel that
  determines the degree of separation between DNA
  molecules of different sizes. In general, linear
  DNA molecules of smaller size percolate through
  the pores/channels of the agarose matrix with
  less drag than the larger molecules

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Problems with Large DNA Molecules

• difficult to handle agarose gels lt 0.7
• large DNA (gt30 kb) migrates via reptation
• reptation results in similar mobilities for large
  molecules
• Reptation    (r?p-t?sh?n)n.1.(Zool.) The act
  of creeping.

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• During continuous field electrophoresis, DNA
  above 30 kb migrates with the same mobility
  regardless of size. This is seen in a gel as a
  single large diffuse band. If, however, the DNA
  is forced to change direction during
  electrophoresis, different sized fragments within
  this diffuse band begin to separate from each
  other.

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• PFGE (Pulsed Field Gel Electrophoresis) is a
  technique developed by Schwartz and co-workers in
  1983 which allows very large (up to 12 megabase)
  DNA fragments to be separated on the basis of
  size

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• - PFGE works by periodically altering the
  electric field orientation
• - the large extended coil DNA fragments are
  forced to change orientation
• - size dependent separation is re-established
  because the time taken for the DNA to reorient is
  size dependent

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If DNA is too large for conventional
electrophoresis.
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Pulsed-field electrophoresis
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Pulse Field Gel Electrophoresis (PFGE)
Electrode configuration of CHEF (contoured-clamp
homogeneous electric field) apparatus
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• direction of electric fields alternated at
  defined intervals
• separation based on ability of DNA to change
  direction
• small molecules reorient faster
• up to 10 Mb can be resolved
• chromosomes of lower eukaryotes
• long-range restriction maps
• in situ lysis of cells and restriction digests

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Preparation of intact DNA for PFGE

• - conventional techniques for DNA purification
  (organic extraction, ethanol precipitation)
  produce shear forces
• - DNA purified is rarely greater than a few
  hundred kb in size
• - this is clearly unsuitable for PFGE which can
  resolve mb DNA
• - at the same time as PFGE itself, techniques to
  purify intact genomic DNA needed to be developed
• - the problem of shear forces was solved by
  performing DNA purification from whole cells
  entirely within a LMT agarose matrix

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• 1) intact cells are mixed with molten LMT agarose
  and set in a mold forming agarose plugs
• 2) enzymes and detergents diffuse into the plugs
  and lyse cells
• 3) proteinase K diffuses into plugs and digests
  proteins
• 4) if necessary restriction digests are performed
  in plugs (extensive washing or PMSF treatment is
  required to remove proteinase K activity)
• 5) plugs are loaded directly onto PFGE and run

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Rare Cutter Restriction Enzymes

• - using the same restriction enzymes as
  conventional molecular biology will result in DNA
  fragments which are far to small for studying
  whole genomes
• - rare cutter restriction endonucleases cut
  genomic DNA with far less frequency than
  conventional REs such as HindIII, BamHI etc.
• - many rare cutter REs have 6-bp (or longer)
  recognition sites eg. NotI GC? GGCCGC
• -

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• suitable rare cutter enzymes have to be
  determined experimentally for each new species
• - in many cases the frequency of cutting is
  highly species dependent eg. BamHI will cut far
  less frequently on a low GC genome when compared
  to a intermediate or high GC content genome

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Applications

• Applications of PFGE are numerous and diverse.
  These include cloning large plant DNAs,
  identifying restriction fragment length
  polymorphisms (RFLP's) and construction of
  physical maps detecting in vivo chromosome
  breakage and degradation and determining the
  number and size of chromosomes ("electrophoretic
  karyotype") from yeasts, fungi, and parasites
  such as Leishmania, Plasmodium, and Trypanosoma.
• PFGE is used to create a DNA "fingerprint" to
  detect and track bacterial strains associated
  with foodborne outbreaks. E. coli 0157H7,
  Shigella, or Salmonella are "fingerprinted" by
  PFGE .

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Saccharomyces cercevisiae chromosomes (245-2190
kb).
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CLONING VECTORS

• In gene cloning, once recombinant DNA is
  constructed, it is introduced into a bacterial or
  eukaryotic host. In the host, recombinant DNA has
  to be maintained, replicated and passed from one
  generation to another. This is achieved by
  introducing recombinant DNA into a cell on a DNA
  vehicle called cloning vector.

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• The most commonly used vectors are plasmid
  cloning vectors.
• Other vector types include bacteriophages,
  cosmids and artificial chromosomes.
• Vector types differ in the molecular properties
  they have and in the maximum size of DNA that can
  be cloned into each.

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Plasmids

• ARTIFICIALLY CONSTRUCTED PLASMIDS USED AS CLONING
  VEHICLES ARE CALLED PLASMID CLONING VECTORS

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MAIN FEATURES OF THE CLONING VECTOR

• Origin of replication
• Selectable Marker
• Multiple cloning site

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• Different types of cloning vectors are used for
  different types of cloning experiments. The
  vector is chosen according to the size and type
  of DNA to be cloned.
• Plasmid vectors are used to clone DNA ranging in
  size from several base pairs to several thousands
  of base pairs.
• Bacteriophage lambda vectors are used to clone
  DNA fragments in the range of 10,000 - 20,000
  base pairs.
• Yeast Artificial Chromosomes (YACs) can be used
  for cloning of hundreds of thousands of base
  pairs.
• Retroviral vectors are used to introduce new or
  altered genes into the genomes of human and
  animal cells.

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A simple cloning exercise
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• pAMP
• 4539 base pairs
• a single replication origin
• a gene (ampr) conferring resistance to the
  antibiotic ampicillin
• a single occurrence of the BamHI restriction
  site
• a single occurrence of the HindIII restriction
  site
• Treatment of pAMP with a mixture of BamHI and
  HindIII produces
• a fragment of 3755 base pairs carrying both the
  ampr gene and the replication origin
• a fragment of 784 base pairs
• both fragments have sticky ends

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Hind III 400
pKAN 4207 bp
Bam HI 2275
KanR
Bgl II 600

• pKAN
• 4207 base pairs
• a single replication origin
• a gene (kanr) conferring resistance to the
  antibiotic kanamycin.
• a single site cut by BamHI
• a single site cut by HindIII
• Treatment of pKAN with a mixture of BamHI and
  HindIII produces
• a fragment of 2332 base pairs
• a fragment of 1875 base pairs with the kanr gene
  (but no origin of replication)
• both fragments have sticky ends

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Extract the fragments for the gel Mix together in
a ligation reaction -ligase -14 C
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pKAN pAMP
Hind III 400
pKAN 4207 bp
Bam HI 2275
KanR
Bgl II 600
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Xba I 200
Hind III ?
Hind III 300
pAMP 4539 bp
AmpR
Bam HI 1184
Bgl II 1600
AmpR
pKAN pAMP ????bp
KanR
Hind III 400
pKAN 4207 bp
Bam HI ?
Bam HI 2275
KanR
Bgl II 600
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Transformation

• Competent cells

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• Competent Cells
• Since DNA is a very hydrophilic molecule, it
  won't normally pass through a bacterial cell's
  membrane. In order to make bacteria take in the
  plasmid, they must first be made "competent" to
  take up DNA. This is done by creating small holes
  in the bacterial cells by suspending them in a
  solution with a high concentration of calcium.
  DNA can then be forced into the cells by
  incubating the cells and the DNA together on ice,
  placing them briefly at 42 C (heat shock), and
  then putting them back on ice. This causes the
  bacteria to take in the DNA. The cells are then
  plated out on antibiotic containing media.

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• Transforming E. coli
• Treatment of E. coli with the mixture of
  religated molecules will produce some colonies
  that are able to grow in the presence of both
  ampicillin and kanamycin.
• A suspension of E. coli is treated with the
  mixture of religated DNA molecules.
• The suspension is spread on the surface of agar
  containing both ampicillin and kanamycin.
• The next day, a few cells resistant to both
  antibiotics will have grown into visible
  colonies containing billions of transformed
  cells.
• Each colony represents a clone of transformed
  cells.

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• Digest with Hind III and Bam HI

3755
2332
1875
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E. coli strains

• E. coli is a commonly used host for propagating
  DNA sequences cloned into plasmid vectors.
  Wild-type E. coli is not a suitable host.
• Cloning E. colis are engineered.
• For example nearly all the strains of E. coli
  carry mutations in RecA

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Phage Cloning Vectors

• Fragments up to 23 kb can be may be accommodated
  by a phage vector
• Lambda is most common phage
• 60 of the genome is needed for lytic pathway.
• Segments of the Lambda DNA is removed and a
  stuffer fragment is put in.
• The stuffer fragment keeps the vector at a
  correct size and carries marker genes that are
  removed when foreign DNA is inserted into the
  vector.
• Example Charon 4A Lambda
• When Charon 4A Lambda is intact,
  beta-galactosidase reacts with X-gal and the
  colonies turn blue.
• When the DNA segment replaces the stuffer region,
  the lac5 gene is missing, which codes for
  beta-galactosidase, no beta-galactosidase is
  formed, and the colonies are white.

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Cosmid Cloning Vectors

• Fragments from 30 to 46 kb can be accommodated by
  a cosmid vector.
• Cosmids combine essential elements of a plasmid
  and Lambda systems.
• Cosmids are extracted from bacteria and mixed
  with restriction endonucleases.
• Cleaved cosmids are mixed with foreign DNA that
  has been cleaved with the same endonuclease.
• Recombinant cosmids are packaged into lambda
  caspids
• Recombinant cosmid is injected into the bacterial
  cell where the rcosmid arranges into a circle and
  replicates as a plasmid. It can be maintained and
  recovered just as plasmids.

Shown above is a 50,000 base-pair long DNA
molecule bound with six EcoRI molecules, and
imaged using the atomic force microscope. This
image clearly indicates the six EcoRI "sites" and
allows an accurate restriction enzyme map of the
cosmid to be generated. http//homer.ornl.gov/cbp
s/afmimaging.htm
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Bacterial Artificial Chromosomes(BACs) and

• BACs can hold up to 300 kbs.
• The F factor of E.coli is capable of handling
  large segments of DNA.
• Recombinant BACs are introduced into E.coli by
  electroportation ( a brief high-voltage current).
  Once in the cell, the rBAC replicates like an F
  factor.
• Example pBAC108L
• Has a set of regulatory genes, OriS, and repE
  which control F-factor replication, and parA and
  parB which limit the number of copies to one or
  two.
• A chloramphenicol resistance gene, and a cloning
  segment.

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Yeast Artificial Chromosomes(YACs)

• YACs can hold up to 500 kbs.
• YACs are designed to replicate as plasmids in
  bacteria when no foreign DNA is present. Once a
  fragment is inserted, YACs are transferred to
  cells, they then replicate as eukaryotic
  chromosomes.
• YACs contain a yeast centromere, two yeast
  telomeres, a bacterial origin of replication, and
  bacterial selectable markers.
• YAC plasmid?Yeast chromosome
• DNA is inserted to a unique restriction site, and
  cleaves the plasmid with another restriction
  endonuclease that removes a fragment of DNA and
  causes the YAC to become linear. Once in the
  cell, the rYAC replicates as a chromosome, also
  replicating the foreign DNA.