Recombinant DNA Technology''

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Recombinant DNA Technology..

• BTEC3301

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DNA Libraries

• How do you identify the gene of interest and
  clone only the DNA sequence you are interested?
  Read Pg 63

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DNA Libraries

• Library screening of gene of interest
• Libraries are collections of cloned DNA fragments
  from a particular organisms contained within
  bacteria or virus as a host.

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DNA Libraries

• Library screening of gene of interest
• Libraries can be kept or saved as such for a long
  time and screened to pick up the gene of interest
  whenever required.

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DNA Libraries

• Library screening of gene of interest
• Two types of libraries which are typically used
  for cloning are genomic DNA libraries and
  complimentary DNA libraries cDNA libraries.

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DNA Libraries

• Human genomic DNA libraries
• Human DNA is cleaved with a restriction enzyme to
  several fragments.
• These fragments are cloned into plasmids

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DNA Libraries

• Human genomic DNA libraries
• Hence a human genomic library consists of a
  collection of bacteria each containing a
  different fragment of human DNA.

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DNA Libraries

• Library screening
• Once the genomic library or cDNA library is
  created, it must be screened for gene of
  interest.

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DNA Libraries

• Library screening
• Although libraries are used for cloning and
  identifying a gene of interest, Polymerase chain
  reaction (PCR) is a much more rapid approach to
  cloning than building a library and screening for
  a gene of interest

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Hybridization

• DNA hybridization
• The pairing of two DNA molecules, often from
  different sources, by hydrogen bonding between
  complementary nucleotides.

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Hybridization

• DNA hybridization
• This term is used to describe the specific
  complementary association due to hydrogen
  bonding, under experimental conditions, of
  single-stranded nucleic acids.

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Hybridization

• DNA hybridization
• It should more properly be referred to as
  "annealing", as this is the physical process
  responsible for the association two
  complementary sequences will form hydrogen bonds
  between their complementary bases (G to C, and A
  to T or U).

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Hybridization

• DNA hybridization
• This technique is frequently used to detect the
  presence of a specific nucleotide sequence in a
  DNA sample.
• Nucleic acid hybridization on membrane filters is
  a simple, sensitive, and specific means of
  detecting nucleic acid sequences of interest.

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Hybridization

• DNA hybridization
• Two types of DNA hybridization
• Southern Blotting
• Northern blotting-RNA

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Hybridization

• DNA hybridization
• Western Blotting is used to analyze proteins
  which have been immobilized on nitrocellulose/nylo
  n filters.

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Hybridization

• DNA hybridization
• Southern blotting (pg75)
• Southern Blot (hybridization) was devised by Ed
  Southern in 1975 for the identification of DNA
  fragments that are complementary to a known DNA
  sequence.

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Hybridization

• DNA hybridization
• Southern hybridization allows a comparison
  between the genome of a particular organism and
  that of an available gene or gene fragment.

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• The Polymerase Chain Reaction (PCR) provides an
  extremely sensitive means of amplifying small
  quantities of DNA.

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Polymerase Chain Reaction (PCR)

• Diagrammatic steps in PCR process (Review)
• Three major steps in PCR
• 1. Template denaturation
• 2. Primer annealing
• 3. Primer extension

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Fig. 3.8 The Polymerase Chain Reaction
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Go to Animation courtesy of the following
websitesReferences

• http//faculty.plattsburgh.edu/donald.slish/PCRmov
  .html (Animation)
• http//www.accessexcellence.org/RC/AB/IE/PCR_Xerox
  ing_DNA.html
• http//www.people.virginia.edu/rjh9u/pcranim.html
  ( PCR Animation)
• http//www.escience.ws/b572/L3/L3.htm (PCR
  Animation)
• http//homepages.strath.ac.uk/dfs99109/BB211/Reco
  mbDNAtechlect4.html
• http//en.wikipedia.org/wiki/Polymerase_chain_reac
  tion
• http//www.escience.ws/b572/L3/L3.htm
• http//allserv.rug.ac.be/avierstr/principles/pcra
  ni.html