Recombinant%20DNA%20Technology%20I

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Recombinant DNA Technology I Lodish Chapters 5.2
it all began with the discovery of the bacterial
defense system that RESTRICTS phage growth. In
the late 1960s, Stewart Linn and Werner Arber
discovered two classes of enzymes methylases and
RESTRICTION endonucleases. At the same time,
Charles Richardson had purified DNA ligase of the
E.coli phage T4.all you needed to do was to
cut and ligate..
And thats what Paul Berg did in the 70sand he
received the Nobel Prize in 1980.
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Restriction enzymes cut DNA molecules at
specific sequences
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Most restriction enzymes recognize short
palindromes and cut unmethylated DNA
Frequency of 6 cutters 46 once every 4096
bp Frequency of 4 cutters 44 once every 256 bp
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Today we know more than 600 different
restriction endonucleases
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DNA cloning with plasmid vectors

• Recombinant DNA technology depends on the ability
  to produce large numbers of identical DNA
  molecules (clones)
• Clones are typically generated by placing a DNA
  fragment of interest into a vector DNA molecule,
  which can replicate in a host cell
• When a single vector containing a single DNA
  fragment is introduced into a host cell, large
  numbers of the fragment are reproduced along with
  the vector

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Plasmid vectors have an ori, a resistance marker
and a multi-cloning site (polylinker)
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Cloning is a 2 step process 1) integrate DNA
fragment into vector
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and 2) transform E.coli to multiply DNA
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Identical E.coli clones carry identical (cloned)
DNA
Usually great to clone short fragment of a few kb
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Complementary DNA (cDNA) libraries are prepared
from isolated mRNAs
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Preparation of a cDNA library
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How do you identify a clone that carries a
specific gene?
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How will I obtain the sequence of my cDNA? The
Sanger (dideoxy) method
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Sample printout from an automated sequencer
N nucleotide cannot be assigned
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Polymerase chain reaction an alternative to
cloning

• The polymerase chain reaction (PCR) can be used
  to amplify rare specific DNA sequences from a
  complex mixture when the ends of the sequence are
  known
• PCR comes in two flavors 1) DNA template based,
  or 2) RNA template based (reverse transcriptase
  PCR)

.Kary Mullis, surfing father of PCR sold the
technology to Cetus for 10,000. Cetus sold the
technology for a stunning 300,000,000 a few
years later Mullis received the Nobel Prize in
1993 and turned his back on both academia and
industry.
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Polymerase chain reaction
thermostable polymerase
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PCR products can be cloned into vectors (e.g.,
for protein expression)
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Reverse transcriptase PCR (can be used in clinic
to probe patient sample for oral pathogens)
Start with single stranded template (mRNA)
RNase H
gene-specific primer
add second gene-specific primer to amplify (by
regular PCR)
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Transposon mutagenesis This strategy is widely
used to create mutants in bacteria and allows for
easy identification of the disrupted gene.
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• What you need to know
• Definition of recombinant DNA (artificially
  ligated DNA fragment)
• Purpose of restriction enzymes (cut DNA,
  naturally used in defense
• against phages)
• Definition of cloning (making lots of identical
  organisms/DNAs)
• Requirements for cloning vectors (3 ori,
  selection marker, polylinker)
• Principal of DNA sequencing (use ddNTPs to
  terminate chain extension)
• Principal of PCR (use thermostable polymerase and
  specific primers to
• amplify a DNA fragment in multiple rounds of
  strand melting, primer
• annealing, primer extension). Principle of
  transposon mutagenesis.
• When to use PCR vs traditional cloning
• DNA sample is limited
• some sequence information is available (there
  are thousands of bugs that
• have NOT been sequenced but play a role in human
  pathologies).
• in general, it is cheaper to create large amounts
  of recomb. DNA by cloning,
• and bacterial enzymes have proofreading
  activities (PCR can create errors)
• Why would you want to know about cDNA cloning?
• gives you information on what sequences are
  expressed (transcribed and
• translated) rather than just encoded in the
  genome