Recombinant DNA Technology……….. BTEC3301. DNA Libraries How do you identify the gene of interest and clone only the DNA sequence you are interested? Read

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  • Recombinant DNA Technology..BTEC3301

  • DNA Libraries

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

  • DNA LibrariesLibrary screening of gene of interest

    Libraries are collections of cloned DNA fragments from a particular organisms contained within bacteria or virus as a host.

  • DNA LibrariesLibrary 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.

  • DNA LibrariesLibrary 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.

  • DNA LibrariesHuman genomic DNA libraries

    Human DNA is cleaved with a restriction enzyme to several fragments.

    These fragments are cloned into plasmids

  • DNA LibrariesHuman genomic DNA libraries

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

  • DNA LibrariesLibrary screening

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

  • DNA LibrariesLibrary 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

  • HybridizationDNA hybridization

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

  • HybridizationDNA 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.

  • HybridizationDNA hybridization

    Western Blotting is used to analyze proteins which have been immobilized on nitrocellulose/nylon filters.

  • HybridizationDNA 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.

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

  • Polymerase Chain Reaction (PCR)Diagrammatic steps in PCR process (Review):

    Three major steps in PCR:

    1.Template denaturation 2.Primer annealing 3.Primer extension

  • Fig. 3.8 The Polymerase Chain Reaction

  • Go to Animation courtesy of the following websites:

    References http://faculty.plattsburgh.edu/donald.slish/PCRmov.html (Animation)http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.htmlhttp://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/RecombDNAtechlect4.htmlhttp://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://www.escience.ws/b572/L3/L3.htmhttp://allserv.rug.ac.be/~avierstr/principles/pcrani.html