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Chapter 5
Exploring genes
Foundation
Enzymology of proteins that alter Nucleic Acids
-Hybridization of DNA with DNA (cDNA) or RNA
-Efficient sequencing methods
-Deliver DNA to host organism
Basic Tools
•  Restriction enzymes;
•  Blotting techniques (Southern, DNA;
Northern, RNA);
•  DNA sequencing;
•  Solid phase synthesis of nucleic acids;
•  The polymerase chain reaction (PCR).
Restriction enzymes
•  Proteins;
•  Restriction endonucleases, recognize specific
DNA sequence and cleave at specific DNA
sequence (scalpels or scissors);
•  Discovered by Werner Arber and Hamilton Smith
and first used by Daniel Nathans;
•  Usually recognize palindromic sequences.
A man a plan a canal panama
“amanap lanac a nalp a nam a”
Sticky
Sticky
Blunt
Sticky
Sticky
Restriction fragments can be separated by
gel electrophoresis and visualized.
Southern Blotting
-DNA is denatured to single strands before being transfered
DNA sequencing: Sanger
dideoxy method
DNA sequencing with fluorescent tags
A complete genome: Haemophilus influenzae
DNA Synthesis: automated solidphase method
The Polymerase Chain Reaction
(PCR)
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Kary Mullis, 1984;
Thermal cycler;
Taq DNA polymerase;
dNTP;
DNA template;
Primers.
Heat to 95°C for 15 s
Heated to 72°C,
optimum for Taq
polymerase
The first cycle in PCR
No new reagents added
PCR
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Strand separation (denaturation, 950C);
Hybridization of primers (annealing; 550C);
DNA synthesis (extension) (720C);
20 to 30 cycles (220 to 230 molecules)
Can be used for detecting
bacteria (tuberculosis) and
viruses (HIV) rapidly.
-Very important for forensics
(blood or hair etc.)and legal
medicine (paternity and
immigration)
DNA and Forensics
Recombinant DNA Technology
•  Pioneered by Paul Berg, Herbert Boyer, and
Stanley Cohen in 1970s;
•  Revolutionized all aspects of biology.
Restriction enzymes and DNA ligase
Vectors for DNA cloning
Plasmids;
λ phage
DNA molecules that can replicate autonomously in an
appropriate host organism.
Specific genes can be cloned from digests of
genomic DNA: genomic library
Detection of DNA with a radiolabelled probe after transfer
from master plate to nitrocellulose and lysis
Introns
Can be a problem for bacteria expression of eukaryotic proteins
Formation of cDNA
Synthesis of proinsulin by bacteria
Proteins with new function can be created
-Deletion of a piece of DNA
-Substitution, single aa
oligonucleotide-directed
mutagenesis.
Half the progeny will
have the mutant and half will not
-Cassette Mutagenesis (see next slide)
Cassette Mutagenesis
Analyzing gene expression in
eukaryotes at the mRNA level
Quantitative PCR (qPCR)
Determining mRNA expression level in cells via cDNA
Levels of Gene Expression
(amount of mRNA)
Test all mRNAs at once
Fluorescently labeled
cDNA is bound to
microarrays of DNA
Microarrays prepared to
complementary to known
sequences obtained from
genomic sequencing
Genes inserted into Eukaryotic cells can be
efficiently expressed.
-Many post translational modifications do not occur in bacteria,
thus eukaryotic genes are not always correctly expressed.
-Provides insight into how genes are organized and expressed.
-Methods for gene introduction include DNA precipitation by
calcium phosphate followed by uptake (low efficiency),
microinjection, or viruses
Microinjection of DNA
Transgeneic mice (growth hormone)
Gene disruption by homologous recombination
(knockouts)
Consequences of gene disruption: sections of muscle from
Normal (A) and gene-disrupted (B) mice, as viewed under
Light microscope. Muscles do not develop properly in mice
Having both myogenin genes disrupted.
Double stranded RNA can inhibit protein
expression