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: Determining DNA sequences

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  1. : Determining DNA sequences Chapter 10: Supplementary lecture

  2. Introduction • determining the base sequence of DNA strands • Obtaining DNA strands: amplification/cloning • PCR (polymerase chain reaction.) • DNA Vectors: Plasmidsand Viruses

  3. Sequencing DNA strands • dATP is an adenine base nucleic acid • ddATP is a modified adenine base which has a coloured florescent marker attached. In has the added property of terminating the elongation if chosen instead of dATP • During the process all possible lengths of chain are produced. • Lengths are separated based on weight and analysed to give • The complementary sequence of the template strand. [ note the sequences in part 1 and part4]

  4. PCR: P. 358 chapter 17: figure 17:10 Klug

  5. How are sequences of genes and genomes obtained • DNA recombinant technology is essential to produce DNA sequences that can be used to determine [chapter 17 (klug 2010)]: • sequences in genes, • regulatory sequences • large DNA strands. • Some of the important terms in this field include: • Cloning DNA: making copies of DNA. • Restriction enzymes: cuts DNA at specific sites : vary in size from sites of 4bp to 8bp or longer; 4 bp cuts into fragments of 256 bp in size ; of 8 b.p 4 8 (64,000) b.p. ; e.g. EcoR1 site: GAATTC • Restriction maps: map of restriction enzyme sites (refer to figure 17.5 klug) Global Sequence

  6. DNA recombinant technology • Plasmid Vectors: help insert the DNA fragment that needs cloned into a host cell. Inside the host cell both the vector and the DNA fragment are cloned (copied). In the example a DNA fragment is inserted into the plasmid. The plasmid is then inserted into the host cells and produces many copies of itself. • The LacZ gene is used as a marker. If markers is disrupted then it means that the host cell has a plasmid vector (recombinant plasmid) in it

  7. Expressed sequence tags • Refer to box 9.1 understanding bioinformatics