Highlights of DNA Technology

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Highlights of DNA Technology
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• Cloning technology has many applications
• Many copies of the gene are made
• Protein products can be produced

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• What is role of
• amp gene in plasmid?
• lacZ gene in plasmid? (see step 2 p 387)
• Hint X-gal product turns blue
• What is the next step after you have identified
  the colonies containing the recombinant plasmid?

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Nucleic Acid Probe Hybridization(p.388-389)
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Then what?

• The colonies of interest are grown in large
  quantities so that the gene of interest can be
  sequenced or so that its product can be produced
• What about the rest of the colonies? (thousands
  may be produced)
• These will be usually be stored and used later or
  shared (or sold) to other scientists (cloning by
  phone)
• A collection of many bacterial clones is a
  genomic library

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Genomic Libraries(can also have phage libraries)
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Polymerase Chain Reaction (PCR)

• a target sequence can be amplified many times
  quickly
• billions of copies after 20 cycles
• Three step cycle denaturation by heating,
  cooling and annealing primers, extension by heat
  stable DNA polymerase

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Gel electrophoresis separates molecules by size
and charge
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RFLP Analysis can be used to detect altered forms
of a gene (polymorphism)
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Southern Blotting of DNA Fragments
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Dideoxy Chain Termination Method for DNA
sequencing
1. Denature strand of DNA
2. Primer added
3. Dideoxynucleotides and normal ones mixed
4. Newly synthesized strands have nucleotides
randomly added
5. dideoxys have fluoresecent or radioactive tags
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Shot gun sequencing
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How can we determine if the gene is functional?

• What do we mean by that?
• Does it produce a product?
• Do different cells produce different products?
• How can we tell the difference?
• How can we tell normal from abnormal cells?
• How we tell the difference between brain cells
  and liver cells?

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Microassays for gene expression
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Short-tandem repeats

• A short tandem repeat (STR) in DNA is a class of
  polymorphism that occurs when a pattern of two or
  more nucleotides are repeated and the repeated
  sequences are directly adjacent to each other.
• The pattern can range in length from 2 to 10 base
  pairs (bp) (for example, CATGCATG) and is
  typically in the non-coding intron region, making
  it junk DNA.

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STRs (cont)

• An individual inherits one marker from one parent
  and the other from the other parent
• By examining several STR loci and counting how
  many repeats of a specific STR sequence there are
  at a given locus, it is possible to create a
  unique genetic profile of an individual.
• STR analysis has become the prevalent analysis
  method for determining genetic profiles in
  forensic cases.

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Comparison of three loci among 4 suspectsWho
does the blood stain belong to?
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Sometimes results are not clear cut