Molecular Cloning Methods

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Molecular Cloning Methods

• ReportorYanLing-Wang
• 2007/9/24

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Contents

• Gene Cloning
• The Polymerase Chain Reaction
• Methods of Expression Cloned Genes

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Gene Cloning

• Restriction Endonuclease
• Vectors
• Indetifing a Specific Clone with a Specific probe

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Restriction Endonuclease

• Characteristics
• Recognizing specific DNA sequences(46Bases)
  which display twofold symmetry and cutting DNA at
  the specific sites producing sticky ends or
  blunt ends
• 5--GAATTC-- 3
• 3--CTTAAG--5
• Sequence
• EcoR1 recognition create sticky ends

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The first cloning experiment involving a
recombinant DNA assembled in vitro
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Question

• If Restriction Endonuclease can cut up invading
  viral DNA, Why they do not destroy host cells
  DNA ?
• Restriction-modification system(R-M system)
• Almost all RE are paired with
• Methylases that recognize and methylate the
  same DNA site ,After methylation,DNA sites are
  protected against RE.
• What about DNA replicaton?

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Vector

• Plasmids as vector (pBR322 )
• Phage as vector (?Phage Vectors )
• Cosmids
• M13 Phage Vector
• Phagemids(pBS)
• Eukaryotic Vectors and Very High Capacity Vectors

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pBR322

• Containing two genes that confer resistance to
  two antibioticsampicillin and tetracycline
• The origin of replication
• EcoRI,BamHI,PstI,HindIII,SalI

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Cloning foreign DNA using PstI site of pBR322

• Cut both plasmids and the insert with
  Pst1,then join them through sticky ends with DNA
  ligase. Next, transform bacteria with the
  recombinant DNA and screen for Tetr and Amps.

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• Questions
• How to find the clones that have received
  recombinant DNAs? (one way is Replica plating)

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Replica plating
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?Phage Vectors

• Accommodating much more foreign DNA
• constructing genomic libraries
• A minimum size requirement for their inserts.
• Charon 4 can accept 12 to 20kb of DNA, a limit
  imposed by the capacity of the ?Phage head.

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Cloning in Charon 4
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Cosmids

• Containing the cos sites of ?phage DNA, which
  allow the DNA to be packaged into ?phage heads
• Containing a plasmid origin of replication,
  which can replicate as plasmids in bacteria.
• The entire ? genome except for the cos sites has
  been removed, they have room for large inserts
  (40-50kb) .

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M13 Phage Vectors

• Mutiple Cloning Sites(MCS)
• Producing single-stranded recombinant DNA, which
  can be used for DNA sequencing and for
  site-directed mutagenesis.

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Obtaining single-stranded DNA by cloning in M13
phage
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Phagemids

• Producing single-stranded DNA.
• Characteristics of both phages and plasmids

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How to identify a Specific Clone with a Specific
Probe?

• Methods
• Polynucleotide probes and antibody probes
• To probe for the cloned gene with polynucleotide
  probes
• 1.using the homologous gene from another organism
  if someone has already cloned

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• 2.Without homologous gene ,how to design the
  sequences of probe?
• If knowing at least part of amino acid sequences
  of protein product of the cloned gene,we can
  deduce the nucleotide sequences by using genetic
  code.

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The polymerase chain reaction

• PCR
• cDNA Cloning
• Reverse transcriptase PCR(RT-PCR)
• Rapid Amplification of cDNA Ends(RACE)

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PCR

• It has three stepsheating,anealling and
  enlongation

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Making a cDNA library
Nick translation
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Nick translation

• It illustrates a generic example with
  double-stranded DNA.
• It can be found that stimultanous degradation of
  DNA ahead of nick and synthesis of DNA behind
  nick
• The principles apply to an RNA-DNA hybrid.

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Nick
Bind E.coli DNA polymeraseI
Simultanous degradation of DNA ahead of nick and
synthesis of DNA behind nick
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RT-PCR

• It is a useful technique for a single cDNA
  cloning.
• The main difference between RT-PCR and PCR is
  that RT-PCR starts with an mRNA instead of a
  double-stranded DNA.

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Using RT-PCR to clone a single cDNA
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RACE

• It is a technique to fill in the missing pieces
  of a cDNA.
• 5 RACE
• 3 RACE

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5 RACE
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Methods of Expressing Cloned Genes

• Expression Vectors
• Other Eukaryotic Vectors
• Using the Ti Plasmid to Transfer Genes to Plants

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Expression Vectors

• Bacterial Expression vectors
• Inducible Expression vectors
• Expression vectors that produce fusion proteins
• Eukaryotic Expression systems

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Oligohistidine expression vector and ?gt11 vector

• Oligohistidine expression vector
• It has a short sequence just upstream of the
  MCS that encode a stretch of six histidines.
• ?gt11 vector
• It contains the lac control region followed by
  the lacZ gene,the cloning sites are located
  within the lacZ gene,so the products of a gene
  inserted correctly intothis vector will be fusion
  protein with a leader of galactosidase

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Question

• Why to attach a stretch of six histidines to a
  protein ?
• Because oligohistidine regions like this have a
  high affinity for divalent metal ions like
  nickel,so protein can be purified by using nickel
  affinity chromatography.

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Expression vectors that produce fusion proteins
Using an oligohistidine expression vector
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Synthesizing a fusion protein in ?gt11
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Why to use Eukaryotic Expression systems?

• Some eukaryotic genes can be expressed in
  prokaryotic system,but it may exist some
  questions.
• 1.the products of the gene can be destoried in
  bacterial cells
• 2.prokaryotes do not carry out the same kinds of
  posttranslational modifications as eukaryotes do.
• 3.The interior of a bacterial cell is not as
  conductive to proper folding of eukaryotic
  proteins as the interior of a eukaryotic cell.

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Eukaryotic Expression systems

• A shuttle vector (prokaryote and eukaryote)
• Abaculovirus vectors

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Expressing a gene in abaculovirus
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Other Eukaryotic Vectors

• Yeast artificial chromosomes (YACs)
• Bacterial artificial chromosomes(BACs)
• PI phage artifial chromosomes(PACs)

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Using the Ti Plasmid to Transfer Genes to Plants

• Ti plasmid
• It can inhabits the bacterium Agrobacterium
  tumefaciens.when the bacteria infects the
  plant,it can transfer Ti plasmid to the host
  cells,whereupon the T-DNA integrates into the
  plant DNA.
• It hasT-DNA gene which has a strong promotor

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Using a T-DNA plasmid to introduce a gene into
tobacco plants
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• Thank you!