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: Determining DNA sequences. Chapter 10: Supplementary lecture. Introduction. determining the base sequence of DNA strands Obtaining DNA strands: amplification/cloning PCR (polymerase chain reaction.) DNA Vectors: Plasmids and Viruses. Sequencing DNA strands.

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determining dna sequences

: Determining DNA sequences

Chapter 10: Supplementary lecture

introduction
Introduction
  • determining the base sequence of DNA strands
  • Obtaining DNA strands: amplification/cloning
    • PCR (polymerase chain reaction.)
    • DNA Vectors: Plasmidsand Viruses
sequencing dna strands
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]
how are sequences of genes and genomes obtained
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

dna recombinant technology
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
expressed sequence tags
Expressed sequence tags
  • Refer to box 9.1 understanding bioinformatics