Chapter 20: DNA Technology and Genomics

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Chapter 20DNA Technologyand Genomics
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Important Point
If you are having trouble understanding lecture
material Try reading your text before
attending lectures. And take the time to read it
well!
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DNA Technology

• DNA technology is the chemical manipulation of
  the genotypes and resulting phenotypes of
  organisms such that living organisms are modified
• Alternatively, no-longer-living organisms or
  their no-longer-living parts may be analyzed
  chemically at the level of genotype
• DNA technology has revolutionized how scientists
  study the genetics, biochemistry, even the
  ecology and evolutionary biology of organisms
• Genetic engineering is the artificial
  manipulation of the genetic material of
  organisms, including the creation of novel
  genetic material (i.e., novel nucleotide
  sequences)
• Biotechnology is the development of novel
  biological products, indeed whole industries are
  now devoted to the production and analysis of
  biological materials

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Cloning Step in Overview
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Have Transformant, then What?

• Clone Identification
• Probing for correct DNA sequence
• Probing for correct protein product
• Antibiotic resistance
• ß-galactocidase expression
• Etc.
• Once you have the correct clone, then what?
• Subcloning
• Expression vector
• Protein characterization
• Protein purification
• Etc.

The final and most difficult part of cloning a
particular gene is identifying a colony
containing that gene among the many thousands of
colonies carrying other pieces of DNA. p. 388,
Campbell Reece (2005)
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Utilizing Cloned Genes
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Restriction Enzymes
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Restriction Endonucleases

• A Restriction Endonucleases will cut both strands
  of a DNA duplex at a specific place
• These places need not be directly opposite

5GAATTC3
3CTTAAG5
5G -OH P-AATTC3
3CTTAA -P HO-G5

• Note that the above enzyme is EcoRI, the first
  restriction endonuclease characterized

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Sticky Ends
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Most R.E. RecognitionSequences are Palindromes
GAATT-C C-TTAAG
GGATC-C C-CTAGG
Nodeba Bob Abedon
AGATC-C T-CTAGG
GCGGCC-GC CG-CCGGCG
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More RE Enzymes
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DNA Ligase
Upon ligation we now have recombinant DNA
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Ligation
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Cloning using Restriction Enzymes
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Transformation
Note that plasmid is vector that carries DNA into
recipient cells via transformation
Other vectors include viruses (transduction) as
well as otherwise inert projectiles
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Gene Therapy
One example of this might be done is essentially
cloning into animals
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Recombinant Animals
Alternatively, DNA can be cloned directly into
the germ line
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Injecting DNA into Egg
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Cloning into Plants
Many plants are easily cloned from individual
body cells
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Selection for Specific Clones
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Selection Nucleic Acid Hybridization
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Genomic Library
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Polymerase Chain Reaction
Cloning allows the amplification of genotype
(DNA) as well as phenotype (proteins)
If all you really need is the DNA, then PCR is an
easy way to amplify DNA without cloning
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PCR DNA Amplification
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PCR DNA Amplification
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Complementary (c)DNA
Rather than making cDNA, one can clone directly
into eukaryotic cells, which addresses concerns
that bacteria do not modify proteins
post-translationally to the extent that
eukaryotes do
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Gel Electrophoresis
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Loading Gel
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Loaded Gel
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Run Gel
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2-D Protein Electrophoresis
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2-D Protein Electrophoresis
Visualized proteins
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Restriction Fragment Analysis
No need to know details of what gene was studied
or specific restriction enzyme used
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Southern Blotting
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Genomic Mapping
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DNA Sequencing
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DNA Sequencing
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DNA Sequencing
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DNA Sequencer
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Shotgun Sequencing
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Genome Comparisons
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Microarray Analysis
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Online Tools

• Buying primers (custom oligos)
  http//www.qiagen.com/
• 2x2 sequence comparisons http//www2.igh.cnrs.fr/
  bin/align-guess.cgi
• n x n sequence comparisons http//www.genebee.msu
  .su/services/malign_reduced.html
• Sequence manipulation http//arbl.cvmbs.colostate
  .edu/molkit/manip/index.html
• Sequence translation http//arbl.cvmbs.colostate.
  edu/molkit/translate/index.html
• Restriction Enzymes http//rebase.neb.com/rebase/
  rebase.html
• Restriction sites http//www.ccsi.com/firstmarket
  /cutter/cut2.html
• Sequence searches http//www.ncbi.nlm.nih.gov/bla
  st/
• Other tools http//molbiol-tools.ca/

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The End