Applications of DNA technology

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Applications of DNA technology

• Human Genome Project
• Human Therapeutics
• Forensic Uses
• Agriculture

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History

• Late 1980s idea was proposed
• Predicted it would take 15 years
• Cost about 200 million per year
• 1 per base pair
• Officially began in 1990
• 26 June 2000 joint announcement from Blair and
  Clinton the draft complete
• Joint publication in Nature and Science 12 Feb
  2001
• 14 Apr 2003 The finished human genome

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Why?

• If I were to study the carburettor of my car
  engine, even in exquisite detail, I would still
  have no idea about the overall function of the
  engine, much less the entire car. To understand
  what an engine is for, and how it works, Id need
  to study the whole thing Id need to place the
  carburettor in context, as one function part
  among many. The same is true of genes..
• James Watson

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Method

• Genetic Mapping
• Identifies relative positions of genes
• E.g. Gene 2 lies between genes 1 and 3
• Physical Mapping
• Absolute positions of genes on chromosomes
• E.g. Gene 2 is 1 million bp from gene 1
• DNA sequencing
• Actual ATCG combinations

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Polymerase Chain Reaction

• Critical to the HGP was the ability to create
  large amounts of DNA for sequencing.
• PCR is a process to amplify DNA
• DNA is heated to 95oC
• The DNA is denatured causing the two strands to
  separate
• A primer (short length of DNA) binds (or anneals)
  to the template strands after the solution is
  cooled
• Complementary DNA strands form through the
  action of DNA polymerase

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Genetic Mapping

• Genetic mapping utilises cross over frequencies
  between known genetic markers.
• A genetic marker is any sequence of genome that
  shows difference between individuals. It could
  be
• A gene
• A Microsatellite or Short Tandem Repeat

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Microsatellites (STRs)

• Repeating sequences 2 4 nucleotide bases found
  in mostly in introns (junk DNA)
• The number of repeats varies from person to
  person, but follow patterns of Mendalian
  inheritance
• These sequences can be identified using probes.
  A probe is
• A short, single stranded sequence of DNA
• Complementary to DNA base sequence
• Tagged radioactively or using fluorescent dye

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Recombinance / Cross Over Frequency

• What do you remember about linked genes from
  Higher.
• What information do recombinant individuals give
  us about the relative position of genes?

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Physical Mapping

• Use of restriction enzymes
• Restriction enzymes belong to the group
  endonucleases or nucleases.
• They cut DNA at specific sequences.
• Chromosomes can therefore be cut into sequences
  of different lengths.
• By using combinations of restriction enzymes and
  working out the size of the fragments, a pattern
  of recognition sites in the DNA can be pieced
  together.

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Gel electrophoresis

• Uses physical properties of DNA (size and charge)
  to separate molecules
• Gel electrophoresis involves running an electric
  current through an agarose gel. DNA is loaded
  into wells at the negative end.
• DNA is repelled, and moves through the agarose
  gel at different speeds depending on the size of
  a fragment.
• A Molecular weight marker (of known fragment
  size) is also used. Samples can be compared to
  this marker to work out their size.

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Physical Mapping cont

• M molecular weight marker
• 1 undigested sample
• 2 digested with NotI
• 3 digested with BamHI
• 4 digested with NotI and BamHI
• 5 something totally different

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Physical Mapping cont
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Physical Mapping cont.
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DNA Sequencing

• Sanger method (aka dideoxy chain-termination
  method)
• An unknown DNA template strand is replicated
  using a primer (to initiate replication), DNA
  polymerase, nucleotide bases and one of 4 dideoxy
  nucleotide bases (ddA, ddG, ddC and ddT)

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DNA sequencing cont

• If a ddNTP is inserted instead of a normal
  nucleotide DNA replication stops at that point
  (chain termination)
• When this experiment is repeated with other dNTPs
  DNA molecules differing in length by 1 bp are
  created.

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DNA sequencing cont..

• These are then run on an electrophoresis gel
• Note The smallest pieces at the start of the
  sequence are at the bottom. Therefore the
  sequence is read bottom to top

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DNA sequencing cont
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Comparing Genomes

• Other genomes have been sequenced in conjunction
  with the human genome, and have continued.
• There are a large number of similarities between
  genomes.
• Genes found in a wide range of organisms are
  known as homologous genes.
• These are used to study function of genes.

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Learning Activities

• Look at the arrangements document to clarify what
  information is required.
• Read DART pg 73 81.
• Read the Monograph pg 67 79
• Scholar 8
• Internet research
• Worksheets