Genetic engineering 2

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  • Genetic Engineering 2
    • DNA sequencing
      • 1. identify difference between normal and mutated genes 2. Confirm sequence of engineered dna 3. Predict the function on a dna sequence
      • Sanger or chain termination method
        • dna to be sequenced used as a template for dna synthesis in vitro (single stranded). Requires a short single stranded piece of dna (15-25bp) to act as starting point (primer) so initial sequence needs to be known
        • Normal nucleotides for dna synthesis are spiked with terminator nucleotides at 100:1. Can be incorporated in the dna strand but prevent subsequent addition of nucleotides.
          • normal dNTP has an OH group at carbon 3. Terminator ddNTP lacks an OH group at carbon 3. This is fluorescently labelled. OH group required for formation of phosphodiester bond between 2 nucleotides.
          • Electrophoresis uses laser to activate fluorescent nucleotides and detector to distinguish colours
    • Current technologies
      • Deep sequencing refers to number of times a nucleotide is read during the sequencing process. Indicates that  the total number of reads is many times larger than the length of the sequence
        • Coverage is average number of reads
      • High throughput - uses lots  of technology to quickly conduct lots of lists to detect active compounds (antibodies or genes that activate a particular biochemistry pathway). Can sequence whole genome in a day
    • Fragmentation of dna by restriction enzymes
      • Purpose; enables analysis of dna and engineering
        • Produces different dna ends (blunt or cohesive (overhanging sticky) ends. Compatible cohesive ends can ligate to form recombinant molecule
      • Restriction enzymes; precise cutters of dna that cleave dna at specific sites. Isolated from bacteria. Thought to protect bacteria from bacteriophages. Own dna protected by methylation or masking of its own restriction sites
      • Characteristics of restriction enzymes; name derived from source e.g. EcoR1 (isolated from ecoli), cut double stranded dna
      • Restriction sites; short usually 4 - 8 bp. Palindromic = sequence of anti-sense (complimentary strand) is the same as that of the sense when read in same orientation
        • Frequency; 4 base site occurs every 256 bases (frequent cutters). 6 base site occurs every 4096. 8 base site occurs every 65536. Rare cutters


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