6'1 Biotechnological Tools and Techniques

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6.1Biotechnological Tools and Techniques
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• A molecular biologist will cut up and join
  segments of DNA to form recombinant DNA
• Recombinant DNA fragment of DNA composed of
  sequences originating from at least two different
  sources.
• in recombinant DNA technology, biologists analyze
  and alter genes and their respective proteins
  with the help of certain tools and techniques

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Restriction Endonucleases

• Restriction Endonuclease enzymes that are able
  to cleave double-stranded DNA into fragments at
  specific sequences also known as restriction
  enzymes
• this is a common tool for molecular biologists
• each type of restriction enzyme recognizes a
  nucleotide sequence known as its recognition site

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• most recognition sites are 4 8 base pairs long,
  usually a palindromic sequence (reads the same
  forwards and backwards)
• Ex. EcoRI bind site
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• G A G A T G A A T T C A G A T
• C T C T A C T T A A G T C T A

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• EcoRI (a restriction enzyme) scans the DNA strand
  looking for its recognition site.
• Once it finds it, it binds to it and disrupts the
  phosphodiester bond (b/w phosphate and sugar)
  through a hydrolysis reaction

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• The ends of the cut DNA differ depending on the
  restriction enzyme that is used.
• EcoRI produces Sticky Ends Fragments of a DNA
  molecule with short single stranded overhangs,
  resulting from cleavage by a restriction enzyme

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Animation for RE

• http//highered.mcgraw-hill.com/classware/ala.do?i
  sbn0072464631alaidala_661286

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• SmaI produces Blunt Ends fragment ends of a DNA
  molecule that are fully base paired, resulting
  from cleavage by a restriction enzyme
• Restriction endonucleases that produce sticky
  ends are more useful than those that produce
  blunt ends.
• This is b/c the sticky ends form H-bonds with
  the complimentary base pairs and require less
  work for repair (enzyme is still required for
  phosphodiester bond)

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• Many tools that are used are biological molecules
  themselves.
• Restriction enzymes are easy to gather b/c they
  are produced in bacteria. They are used as a
  simple defense system.
• The virus DNA is injected into the bacterium and
  the exonuclease scans for recognition sites on
  foreign DNA and cuts them up. Eventually some
  DNA will have a recognition site.

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Methylases

• Restriction endoucleases must be able to
  distinguish between foreign DNA and their own
  cells DNA, if they cant then they will cleave
  their own DNA
• Methylase enzymes that add a methyl group to
  one of the nucleotides found in a restriction
  endonuclease recognition site, altering its
  chemical composition.

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• In prokaryotes, methylase modifies the
  recognition site of a respective restrictive
  endonuclease by placing a methyl group on one of
  the bases, this prevents the endonuclease from
  cutting the DNA.
• Foreign DNA is not methylated and will be
  recognized by the R.E.
• These are important for molecular biologist so
  they can manipulate DNA and protect the area they
  do not want to be cleaved

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DNA Ligase

• After genes are cut they need a way to be
  reconnected
• 2 sticky ends will naturally attract each other
  and form hydrogen bonds, but this is too weak for
  good stability
• DNA ligase will reform the phosphodiester bonds
  hydrolysis reaction

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• To reform the bond between 2 bunt ends molecular
  biologists must use T4 DNA ligase an enzyme
  that originated from the T4 bacteriophage, it is
  used to join blunt ends together.