Download Trends in Biotechnology

Trends in Biotechnology
Chapter 3a – Tools for
Recombinant DNA
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Chapter 3 - outline
II.
III.
Cutting and Joining DNA
Separating Restriction Fragments and Visualizing
DNA
DNA Cloning
Cloning Vectors
IV.
V.
A.
Bacterial Vectors
1.
2.
3.
B.
Plasmids
Bacteriophage
Cosmids
Vectors for Other Organisms
1.
2.
3.
4.
Yeast Artificial Chromosomes (YACs)
Bacterial Artificial Chromosomes (BACs)
Plant Cloning Vectors
Mammalian Cell Vectors
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VI.
VII.
Cell Transformation
Constructing and Screening a DNA
Library
A.
B.
C.
D.
VIII.
Genomic Library
cDNA Library
Screening Libraries
Expression Libraries
Reporter Genes
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IX.
X.
XI.
XII.
XIII.
A.
B.
C.
Southern Blot Hybridization
Northern Blot Hybridization
Polymerase Chain Reaction
DNA Sequencing
Protein Methods
Protein Gel Electrophoresis
Protein Engineering
Protein Sequencing
XIV.
DNA Microarray Technology
A.
Biotech Revolution: RNA Interference
Technology: Gene Silencing
XV.
Applications of Recombinant DNA
Technology
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Learning Objectives
1. Know how restriction endonucleases
work to cut DNA, and why they are
important in biotechnology. Compare
blunt ends with sticky ends.
2. Know how electrophoresis separates
pieces of DNA.
3. List and know the steps of DNA
cloning.
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4. Know how vectors are used to
transform bacteria, and how to select
for successfully transformed bacteria.
Compare the how different vectors can
carry different sizes of DNA into the
bacteria.
5. List the types of vectors that can be
used to transform yeast, mammalian
cells and plants, and why they are
effective in those organisms.
6. List the methods of transformation of
cells.
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7. Compare genomic libraries, cDNA
libraries, and expression libraries. How
are they constructed? What are the
libraries looking for? How they are
screened?
8. List the various types of reporter
genes used in research.
9. Compare Northern and Southern blot
hybridization. How are they constructed?
What is each type of hybridization
looking for?
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10. Know the function of PCR, the steps
of PCR, and what researchers do with
PCR.
11. Compare the two methods of DNA
sequencing: the chemical method and
the Sanger method, and know which
method is more widely used. How does
automation change DNA sequencing?
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12. List and define the various methods
of analyzing proteins. Are any of these
methods similar to DNA methods?
13. Know the types of microarrays, and
how DNA and protein microarrays work.
14. List the applications of recombinant
DNA technology.
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Fig. 3.1 Cutting double-stranded DNA.
(a) The enzyme DNase cuts DNA at
random sites.
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Fig. 3.1 (b) Restriciton enzymes
cut DNA at specific sites.
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Figure 3 a:
Restriction
enzymes
make doublestranded cuts
in the sugarphosphate
backbone of
DNA
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http://www.nature.com/scitable/resource?action=showFullImageForTopic&imgSrc=44956/pierce_18_2_FULL.jpg
Video: Restriction
http://www.dnalc.org/resources/animatio
ns/restriction.html
(13 restriction.exe)
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Fig. 3.1 (c) A Specific restriction
enzyme cuts each DNA molecular at
the same sequence site.
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Fig. 3.2 The ligation of two different pieces of DNA.
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Figure 3 b:
Restriction
enzymes
producing
cohesive, or
sticky, ends.
These ends
can be joined
again.
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http://www.nature.com/scitable/resource?action=showFullImageForTopic&imgSrc=44956/pierce_18_2_FULL.jpg
Video: restriction enzymes bio37
http://highered.mcgrawhill.com/olc/dl/120078/bio37.swf
(13 restriction)
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Fig. 3.3 Agarose gel electrophoresis is used to
separate DNA (and RNA) molecules according to size.
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Electrophoresis:
http://www.dnalc.org/resources/animatio
ns/gelelectrophoresis.html
(20 Electrophoresis - gelelectrophoresis.exe)
Together, restriction enzymes and gel
electrophoresis can give a lot of
information.
http://www.dnalc.org/view/16529Animation-24-The-RNA-message-issometimes-edited-.html
(20 Electrophoresis - rest enzy and electro.exe)
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