Determine nucleic acid sequence

1
Site Directed Mutagenesis to Understand Gene
Function

• Determine nucleic acid sequence
• Determine sequence change required
• Place a synthetic oligonucleotide with the
  corresponding change in association with the DNA
• Semiconservative replication with new
  sequence from oligo
• Transcribe and translate - mutant protein

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Site Directed Mutagenesis
3
Site Directed Mutagenesis
4
Knockouts and Transgenes

• Site directed mutagenesis can study function
  in an in vitro system
• Delivery of that mutated form of the protein
  into a living organism can be used to assess the
  in vivo function
• Deletion of the gene gives a knockout
  organism
• Insertion of a gene in addition to others
  already present will give a transgenic organism

5
Making Knockouts
6
Making Transgenic Organisms
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Transgenic Mice
8
Transposable Elements Get Around

• Mobile genetic elements that can insert and
  excise randomly within a genome
• Often disrupts gene function by insertion
  into the regulatory or structural part of the
  gene
• Insertion and excision are dependant on
  specific sequences in the transposon
• Insertion is dependant on insertions
  sequences (IS) which vary in length

9
Insertion Sequences

• lt 2000 bp in size generally around 1000 bp
• Inverted terminal repeats (ITR) - the 5-3
  sequence is the same as the 5-3sequence on the
  other strand
• Internal sequences (IS) are not well
  conserved
• ITR may allow IS elements to insert at
  specific sites along the chromosome based on
  recognition

10
Transposons

• A collection of IS elements may form a
  mobile element containing genes unrelated to
  insertion from a particular organism
• The transposons can carry information
  similar to a plasmid but can insert more readily
  into the genome
• Example - Tn5 from bacteria has a neomycin
  resistance gene present which many molecular
  biologist have used for selection of transformed
  cells
• Transposons have been found in many
  different organisms including Maize, Yeast,
  Drosophila and Humans

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Transposons
12
P-Element Transposons
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P-Element Transposons
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Transposons Effect Genomic Structure
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Transposons Effect Genomic Structure