DNA Technology

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Chapter 20

• DNA Technology

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DNA Cloning

• Gene cloning allows scientists to work with small
  sections of DNA (single genes) in isolation.
• Exactly what does the gene code for?
• Much of a DNA molecule is noncoding, and
  scientists are mostly interested in the genes.
• Cloning makes identical copies of the same gene
  (or genes)

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Figure 20.1 An overview of how bacterial
plasmids are used to clone genes
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Bacterial Plasmids

• Plasmids are small, circular DNA molecules in
  bacteria.
• By inserting genes into plasmids, scientists can
  combine eukaryotic and prokaryotic DNA.
  (Recombinant DNA)
• Bacterial cells continually replicate the foreign
  gene along with their DNA.
• Cloning using plasmids can be used to
• Identify a particular protein a gene makes (ie
  for study)
• Produce large amounts of a particular
  protein/gene (ie for use in medicine)

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Restriction Enzymes

• Also used to make recombinant DNA.
• Specifically cut DNA molecules at precise base
  locations.
• (restriction)

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Making Recombinant DNA (Fig 20.3)
Making Recombinant DNA (Fig 20.3)
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Still Making Recombinant DNA
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Almost Recombinant
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• DNA Technology Files
• Restriction Enzyme Movie
• Cloning Movie

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Why Use Bacteria as vectors?

• Plasmids are easy to use to manipulate which
  genes are expressed in clones.
• 2. Bacteria replicate very quickly and allow you
  to produce a large number of a desired gene.

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Identifying Clones

• Not all of the reproduced bacteria are clones
  carrying the desired gene.
• Two ways to identify which are clones
• Look for the gene
• Look for the protein the gene codes for

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Nucleic Acid Hybridization (find gene)

• If you know the sequence of the cloned gene you
  are looking for, you can make a nucleic acid
  probe with a complementary sequence.
• The probe is radioactively labeled and allowed to
  base pair with the denatured (separated strands)
  DNA.
• The probes H-bond with their complement (cloned
  gene), thus identifying the cloned cells.
• Identified cells are cultured to produce more.

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Figure 20.4 Using a nucleic acid probe to
identify a cloned gene
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Expressing Euk. Proteins in Bacteria

• It is more difficult to get the bacteria to
  translate the proteins because of differences in
  promotor sequences b/t prokaryotes and
  eukaryotes.
• Expression vectors are plasmids that contain the
  promotor sequence just before the restriction
  site.
• This allows the insertion of a eukaryotic gene
  right next to the prokaryotic promotor.

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Expressing Euk. Proteins in Bacteria

• Bacteria also lack the enzymes needed to remove
  introns from DNA.
• Therefore, cDNA (no introns) is inserted into
  plasmids to allow expression of the eukaryotic
  gene.
• Reverse transcriptase is the enzyme used to make
  cDNA from a fully processed mRNA strand.

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Figure 20.5 Making complementary DNA (cDNA) for
a eukaryotic gene
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Another Solution Use Yeast (eukaryotic)

• Why?
• They grow quickly like bacteria
• They are eukaryotes (similar enzymes, metabolic
  mechanisms, protein mods)
• They have plasmids (rare for eukaryotes)
• Can replicate artificial chromosomes as well as
  DNA in plasmids

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Genomic Libraries

• Plasmids and phages used to store copies of
  specific genes.

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Polymerase Chain Reaction (PCR)
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PCR

• Faster and more specific method for amplifying
  short DNA sequences
• After DNA is denatured (split), primers start new
  complementary strands with each strand producing
  more molecules of the sequence.
• In vitro doesnt require living cells
• In test tube denatured DNA, free nucleotides,
  DNA primers (specific to gene desired), special
  DNA polymerase (can withstand high heat w/o
  denaturing)

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Analyzing DNA

• Gel electrophoresis separates molecules based on
  size, charge, density, etc.
• Linear DNA mainly separated by fragment length
  (size)
• Molecules of DNA are separated into bands of
  molecules of the same length.

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Gel Electrophoresis
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Restriction Fragment Analysis
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Southern Blotting
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Southern Blotting

• Produce restriction fragments of DNA (rest.
  enzyme used)
• Separate fragments (gel electrophoresis)
• Blotting
• Transfer DNA to nitrocellulose paper via cap.
  action
• Hybridize with radioactive probes (know seq.)
• Autoradiography to identify which have probes.

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RFLPs (rif-lips)

• Polymorphisms that result from differences in
  noncoding regions of DNA.
• Restriction enzymes cut DNA into different
  fragments in each variant.
• RFLP markers allowed scientists to more
  accurately map the human genome.
• Genetic studies do not have to rely on phenotypic
  (appearance/proteins) differences to guide them
  anymore.

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In Situ (on a slide) Hybridization

• Radioactively (or fluorescently) labeled probes
  base pair with complementary denatured DNA on a
  microscope slide.
• Autoradiography and staining identify the
  location of the bound probe.

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Human Genome Project

• Attempt to map the genes on every human
  chromosome as well as noncoding information.
• Three stages
• Genetic Mapping (linkage)
• Physical Mapping
• Gene (DNA) Sequencing
• Genomes of species that give insight to human
  codes are also being done (fruit fly, E coli,
  yeast)

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Genetic Mapping (Stage 1)

• Linkage maps based on recombination frequencies
  created.
• Linkage maps portray gene sequences as you
  physically move along a chromosome.
• Genetic markers along the chromosome allow
  researchers to use them as reference points while
  studying other genes.

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Physical Mapping (Stage 2)

• Determines the actual distance between the
  markers along a chromosome ( of bases)
• Utilizes chromosome walking to identify the
  distance between.
• Use a series of probes to identify the DNA
  sequence of various restriction fragments, and
  ultimately the entire length of DNA sample.

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Chromosome Walking
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DNA Sequencing (Stage 3)

• As of 1998, 3 of the human genome had been
  sequenced using automation. (Sanger Method)
• Once the sequences of all the genes are known,
  scientists can begin to study all of their
  functions, and manipulate their products in many
  ways.

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Applied Genetics

• Diagnosis of Genetic Disorders
• Sequence individuals before birth to know if
  their DNA contains abnormalities
• Human Gene Therapy
• Replace missing or fix damaged genes in affected
  individuals

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Gene Therapy
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Pharmaceuticals

• Hormone production (ie Human Growth)
• Protein supplements
• HIV treatment decoy receptor protein used to
  inhibit HIV virus ability to enter cell
• Vaccines
• Proteins that stimulate immune response can be
  used instead of traditional vaccines
• Antisense Nucleic Acids
• Block translation of certain proteins

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Other Uses of DNA Tech

• DNA Fingerprinting for forensic cases
• Environmental cleanup
• Agriculture
• Animal Husbandry
• Genetic Engineering of Plants

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The Future of Genetics

• The future of science lies in genetics.
• The question is not whether or not we can do the
  things discussed in this chapter, but whether or
  not we should. This is a question you will
  ultimately have to help answer.