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Chapter 20: DNA Technology and Genomics

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Chapter 20: DNA Technology and Genomics Important Point: DNA Technology Cloning Step in Overview Have Transformant, then What? Utilizing Cloned Genes Restriction ... – PowerPoint PPT presentation

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Title: Chapter 20: DNA Technology and Genomics


1
Chapter 20 DNA Technology and Genomics
2
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!
3
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

4
Cloning Step in Overview
5
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)
6
Utilizing Cloned Genes
7
Restriction Enzymes
8
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

9
Sticky Ends
10
Most R.E. Recognition Sequences are Palindromes
GAATT-C C-TTAAG
GGATC-C C-CTAGG
Nodeba Bob Abedon
AGATC-C T-CTAGG
GCGGCC-GC CG-CCGGCG
11
More RE Enzymes
12
DNA Ligase
Upon ligation we now have recombinant DNA
13
Ligation
14
Cloning using Restriction Enzymes
15
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
16
Gene Therapy
One example of this might be done is essentially
cloning into animals
17
Recombinant Animals
Alternatively, DNA can be cloned directly into
the germ line
18
Injecting DNA into Egg
19
Cloning into Plants
Many plants are easily cloned from individual
body cells
20
Selection for Specific Clones
21
Selection Nucleic Acid Hybridization
22
Genomic Library
23
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
24
PCR DNA Amplification
25
PCR DNA Amplification
26
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
27
Gel Electrophoresis
28
Loading Gel
29
Loaded Gel
30
Run Gel
31
2-D Protein Electrophoresis
32
2-D Protein Electrophoresis
Visualized proteins
33
Restriction Fragment Analysis
No need to know details of what gene was studied
or specific restriction enzyme used
34
Southern Blotting
35
Genomic Mapping
36
DNA Sequencing
37
DNA Sequencing
38
DNA Sequencing
39
DNA Sequencer
40
Shotgun Sequencing
41
Genome Comparisons
42
Microarray Analysis
43
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/

44
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45
The End
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