Chapter 20DNA Technology

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Chapter 20 DNA Technology Genomics
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O.J. Simpson capital murder case,1/95-9/95

• Odds of blood in Ford Bronco not being R.
  Goldmans
• 6.5 billion to 1
• Odds of blood on socks in bedroom not being N.
  Brown-Simpsons
• 8.5 billion to 1
• Odds of blood on glove not being from R. Goldman,
  N. Brown-Simpson, and O.J. Simpson
• 21.5 billion to 1
• Number of people on planet earth
• 6.1 billion
• Odds of being struck by lightning in the U.S.
• 2.8 million to 1
• Odds of winning the lottery
• 76 million to 1
• Odds of getting killed driving to the gas station
  to buy a lottery ticket
• 4.5 million to 1
• Odds of seeing 3 albino deer at the same time
• 85 million to 1
• Odds of having quintuplets
• 85 million to 1
• Odds of being struck by a meteorite

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Recombinant DNA

• Def DNA in which genes from 2 different sources
  are linked
• Genetic engineering direct manipulation of
  genes for practical purposes
• Biotechnology manipulation of organisms or their
  components to perform practical tasks or provide
  useful products

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Bacterial plasmids in gene cloning
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DNA Cloning

• Restriction enzymes (endonucleases) in nature,
  these enzymes protect bacteria from intruding
  DNA they cut up the DNA (restriction) very
  specific
• Restriction site recognition sequence for a
  particular restriction enzyme
• Restriction fragments segments of DNA cut by
  restriction enzymes in a reproducable way
• Sticky end short extensions of restriction
  fragments
• DNA ligase enzyme that can join the sticky
  ends of DNA fragments
• Cloning vector DNA molecule that can carry
  foreign DNA into a cell and replicate there
  (usually bacterial plasmids)

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Steps for eukaryotic gene cloning

• Isolation of cloning vector (bacterial plasmid)
  gene-source DNA (gene of interest)
• Insertion of gene-source DNA into the cloning
  vector using the same restriction enzyme bind
  the fragmented DNA with DNA ligase
• Introduction of cloning vector into cells
  (transformation by bacterial cells)
• Cloning of cells (and foreign genes)

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Identifying cell clones with the right gene.

• In the final step, we will sort through the
  thousands of bacterial colonies with foreign DNA
  to find those containing our gene of interest.
• One technique, nucleic acid hybridization,
  depends on base pairing between our gene and a
  complementary sequence, a nucleic acid probe, on
  another nucleic acid molecule
• The probe will hydrogen-bond specifically to
  complementary single strands of the desired
  gene.

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complementary DNA (cDNA)

• The presence of introns, long non-coding regions,
  in eukaryotic genes creates problems for
  expressing these genes in bacteria.
• To express eukaryotic genes in bacteria, a fully
  processed mRNA acts as the template for the
  synthesis of a complementary strand using reverse
  transcriptase
• Complementary DNA is DNA made in vitro using mRNA
  as a template and the enzyme reverse
  transcriptase.

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Techniques to facilitate entry of foreign DNA

• In electroporation, brief electrical pulses
  create a temporary hole in the plasma membrane
  through which DNA can enter.
• Alternatively, scientists can inject DNA into
  individual cells using microscopically thin
  needles.
• In a technique used primarily for plants, DNA is
  attached to microscopic metal particles and fired
  into cells with a gun.
• Once inside the cell, the DNA is incorporated
  into the cells DNA by natural genetic
  recombination

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DNA Analysis Genomics

• PCR (polymerase chain reaction)
• Gel electrophoresis
• Restriction fragment analysis (RFLPs)
• Southern blotting
• DNA sequencing
• Human genome project

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Polymerase chain reaction (PCR)

• Amplification of any piece of DNA without cells
  (in vitro)
• Materials heat, DNA polymerase, nucleotides,
  single-stranded DNA primers
• Applications fossils, forensics, prenatal
  diagnosis, etc.

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DNA Analysis

• Gel electrophoresis separates nucleic acids or
  proteins on the basis of size or electrical
  charge creating DNA bands of the same length

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Restriction fragment analysis I

• Restriction fragment analysis indirectly detects
  certain differences in DNA nucleotide sequences
• Restriction fragment analysis is sensitive enough
  to distinguish between two alleles of a gene that
  differ by only base pair in a restriction site.

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Restriction fragment analysis II

• Restriction fragment length polymorphisms (RFLPs)
  Differences in DNA sequence on homologous
  chromosomes that produce different restriction
  fragment patterns are scattered abundantly
  throughout genomes, can serve as a genetic marker
  for a particular location (locus) in the genome
• Southern blotting process that reveals
  sequences and the RFLPs in a DNA sequence
• DNA Fingerprinting

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DNA Fingerprinting
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DNA Sequencing

• Determination of nucleotide sequences (Sanger
  method, sequencing machine)
• Genomics the study of genomes based on DNA
  sequences
• Human Genome Project

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Practical DNA Technology Uses

• Diagnosis of disease
• Human gene therapy
• Pharmaceutical products (vaccines)
• Forensics
• Animal husbandry (transgenic organisms)
• Genetic engineering in plants
• Ethical concerns?