Biotechnology

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Biotechnology

• The basics, and selected applications

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Some terms

• Recombinant DNA combining DNA from different
  sources into one molecule in vitro
• Biotechnology applied biology ad technology
• Genetic engineering manipulation of genes for a
  specific purpose
• There is nothing new about biotechnology- just
  some of the techniques

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More terms

• Plasmids
• Small circular pieces of DNA
• Found naturally in bacteria
• Replicate independently of the chromosome (often
  in multiple copies)
• DNA fragments can be inserted in them
  expression vector
• Cloned genes multiple copies (and maybe large
  quantities of gene product)

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An overview of cloning
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Recombinant DNA technology

• Restriction endonucleases cleave DNA
• Type II cut at specific sites (palindromes)
• DNA from different species are cut the same way-
  so they can be joined together
• DNA ligase seals the ends
• Many technological advances have contributed to
  our understanding of gene expression

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Other uses for expression vectors
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Vectors come in different sizes

• Plasmid- up to 10-15,000 bp insert
• Viruses- up to 40-50,000 bp
• Artificial chromosomes- over 100,000
• (remember, some eukaryotic genes are huge!)
• Can you recover the information that ends up in
  RNA, without the intervening sequences?

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Yes- cDNA

• cDNA vs genomic DNA
• Are you studying the gene or the protein?
• cDNA fits into bacterial expression vectors
• Genomic DNA will contain regulatory sequences

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How do you find a DNA sequence of interest?
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PCR can amplify a single DNA Molecule Forensic Me
dical Basic research
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Agarose gel electrophoresis allows rapid analysis
of DNA
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RFLP analysis
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You are not always lucky enough to have a RFLP
within a gene!

• SNPs (single nucleotide polymorphisms)
• Humans have lots of them
• They can be markers close to genes of interest

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Microarrays allow you to scan combinations of
expressed genes in different tissues
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DNA sequencing technology has developed rapidly

• Largely automated
• Techniques become faster and cheaper
• Made genomic sequencing possible
• Does not require radioactivity
• Data storage

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Hybridization finding sequences of interest

• Several hybridization techniques have been
  developed
• Southern blots identify genomic sequences
• Northern blots- RNA is gene transcribed?
• Western blots detect proteins in a mixture
• Southwestern blots!

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Southern blotting is a classic method of DNA
analysis
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PCR is replacing many hybridization techniques
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Cloning multicellular organisms

• Origin is a single cell
• Plants are easy to clone
• Nuclear transfer nucleus from what type of cell?
• Undifferentiated successful since 1950s
• Differentiated first achieved in 1997
• Cloning is not problem-free

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Stem cells undifferentiated, or partially
differentiated
Cells can be reprogrammed to be pluripotent
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What is regenerative medicine?
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Applications of recombinant DNA technology

• Expression (and mass production) of proteins in
  microbial cells
• Vaccine development (subunit, DNA)
• Gene therapy
• Agricultural applications
• Resistance to herbicides, insects, frost
• GMOs
• Transgenic animals

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What is the relationship between science and
technology? (adapted from Kreuzer and Massey,
2005)

• Science
• Search for knowledge about ourselves and the
  physical world
• Process scientific method looking for patterns
  in the physical world evaluated by how well
  facts support conclusions limited by ability to
  collect evidence
• Discoveries give rise to technological advances

• Technology
• Practical application of knowledge adapting
  ourselves to physical world
• Process finding solutions to problems try to
  control physical world evaluate by how well it
  works limited by costs and available technology
• Advances give rise to scientific discoveries

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Summary

• Science drives technology, and technology drives
  science.
• Biotechnology has been around as long as humans
  have practiced agriculture.
• Biotechnology is used to conduct research,
  develop new products, and improve processes.
• DNA-based technologies have been developed in
  microbes- and beyond.
• As a society, we are responsible for using it
  wisely and ethically.