Introduction to biotechnology

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Introduction to biotechnology

• Haixu Tang
• School of Informatics

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Biotechnology

• Cell technology
• Isolating cells
• Growing cells in culture
• Fractionating cells
• Molecular technology
• DNA cloning
• DNA sequencing
• Gene expression
• Analyzing protein functions

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Isolating Cells

• Disrupting extracellular matrix proteolytic
  enzymes or EDTA
• Separating different cell types
• Antibodies coupled to a fluorescent dye
  (fluorescence-activated cell sorter)
• Microscopic dissection
• Cell growth

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Fluorescence-activated cell sorter
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Microscopic dissection
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Growing cells in culture
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Composition of a Typical Medium Suitable for the
Cultivation of Mammalian Cells
AMINO ACIDS VITAMINS SALTS MISCELLANEOUS PROTEINS
Arginine biotin NaCl glucose insulin
Cystine choline KCl penicillin transferrin
Glutamine folate NaH2PO4 streptomycin growth factors
Histidine nicotinamide NaHCO3 phenol red
Isoleucine pantothenate CaCl2 whole serum
Leucine pyridoxal MgCl2
Lysine thiamine
Methionine riboflavin
Phenylalanine
Threonine
Trytophan
Tyrosine
Valine
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Cell line

• Most vertebrate cells stop dividing after a
  finite number of cell divisions in culture
  senescence
• "immortalized" cell line telemerase
• Inactivate the checkpoint mechanisms
• Cell lines can often be most easily generated
  from cancer cells.

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Hybrid cells

• a heterocaryon, a combined cell with two separate
  nuclei

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Ultracentrifuge
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Cell fractionation by centrifugation
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velocity sedimentation vs. equilibrium
sedimentation
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Column chromatography
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Matrices used for chromatography
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Protein purification by chromatography
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SDS polyacrylamide-gel electrophoresis
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Western blotting
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Protein identification via MS
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DNA recombination technology

• 1. Cleavage of DNA at specific sites by
  restriction nucleases, which greatly facilitates
  the isolation and manipulation of individual
  genes. 2. DNA cloning either through the use of
  cloning vectors or the polymerase chain reaction,
  whereby a single DNA molecule can be copied to
  generate many billions of identical molecules.
  3. Nucleic acid hybridization, which makes it
  possible to find a specific sequence of DNA or
  RNA with great accuracy and sensitivity on the
  basis of its ability to bind a complementary
  nucleic acid sequence. 4. Rapid sequencing of
  all the nucleotides in a purified DNA fragment,
  which makes it possible to identify genes and to
  deduce the amino acid sequence of the proteins
  they encode. 5. Simultaneous monitoring of the
  expression level of each gene in a cell, using
  nucleic acid microarrays that allow tens of
  thousands of hybridization reactions to be
  performed simultaneously.

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Restriction nucleases
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Restriction nucleases produce DNA fragments that
can be easily joined together
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DNA Gel electrophoresis
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DNA hydridization
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Gel transferred hybridization
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In situ hydridization
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In situ hybridization for RNA localization
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DNA recombination
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DNA cloning
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YAC
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Human genome library
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The synthesis of cDNA
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cDNA clones and genomic DNA clones
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PCR
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PCR based gene cloning
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PCR used in forensic science
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Protein expression systemvia a plasmid
expression vector
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Molecular biology research
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Studying gene functions

• Genetic screening
• Monitoring gene expression
• Site-directed mutagenesis
• Gene replacement or knockout

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LDA
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Reporter gene
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Site-directed Mutagenesis
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Genome manipulation
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Anti-sense RNA strategy
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Making collections of mutant organisms
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Mouse with an engineered defect in fibroblast
growth factor 5 (FGF5)