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Survey ofmolecular techniques
Enzymes for DNA manipulation
DNA polymerases
nucleases
ligases
end-modification enzymes
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DNA polymerases(template-dependent)
synthesize new DNA chains (complementary to template)
require oligonucleotide primers
may possess 3-5 exonuclease activity (for proofreading)
some forms are thermostable (e.g., Taq polymerase)
some forms require RNA as template (RNA polymerases),
synthesizing cDNAs
Nucleases
hydrolyze phosphodiester bonds in nucleic acids
some are specific for DNA, others for RNA
main types:
exonuclease
endonuclease
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Exonucleases: examples
Exonuclease III - removes residues from 3 ends
of a DNA strand
Bacteriophage exonuclease - removes residues
from 5 ends of a DNA strand
Endonucleases
cut at internal phosphodiester bonds
restriction enzymes - an important class ofDNA endonucleases; play a central role in
recombinant DNA technology
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Restriction enzymes
Molecular scissors that cut double stranded
DNA at defined sites
3 classes
Type II restriction enzymes are most commonly
used; cut DNA at defined sites
Nomenclature
Each restriction enzyme is named after thebacteria it was isolated from
For example:EcoRI (pronounced eeko-are-one)
E = Genus, Escherichia
co = Species, coli
R = Strain, RY13
I = First endonuclease isolated fromthis bacteria
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Restriction Sites
Specific sequence of nucleotides that aparticular restriction enzyme recognizes
Most restriction sites are about 4-6 baseslong
Many are palindromic
- GGATCC -
- CCTAGG -
Frequency that a restriction site is likely tooccur in a strand of DNA can be predicted
1/4n where n = # of bases in the restriction siteA four base pair restriction site will occur every
256 bases
Types of ends after restriction
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Restriction enzymes: examples
EcoRI GAATTC sticky (4bp)
HindIII AAGCTT sticky (4bp)
Sau3A GATC sticky (4bp)
PvuII CAGCTG blunt
HaeIII GGCC blunt
NotI GCGGCCGC sticky (4bp)
HinfI GANTC sticky (3bp)
DNA ligases
catalyze joining of two DNA strands by
synthesizing a phosphodiester bond
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5
3
G
C T T A A
A A T T C
G
one DNA fragment another DNA fragment
Ligation of DNA fragments
annealing of sticky ends
G A A T T C
C T T A A G3
5 3
5
G A A T T C
C T T A A G
DNA ligase-catalyzed ligation
+
Ligation of DNA fragments
Ligation of joining of double strands vs.
ligation of nicked fragments
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Recombinant DNA Technology
Involves the combination of DNA from two different
sources Allows researchers to artificially manipulate genetic
material; sequences are cut with restriction enzymes
and joined together with ligases
Revolutionized the way DNA sequences were studied
Produce proteins
Study gene expression
Gene function
Recombinant Plasmid
++
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End-modification enzymes
terminal transferase
adds homopolymer tails to the 3 ends of a linear
duplex
T4 polynucleotide kinase
adds a phosphate group to the 5-OH end of a
polynucleotide (to label it or to permit ligation)
alkaline phosphataseremoves phosphate group from 5 ends
Polymerase Chain Reaction
(PCR)
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Polymerase Chain Reaction (PCR)
94 oC
1 min 50 oC
20 s
72 oC
1 min
denaturation
annealing
of primers
extension
DNA
Parameters of a PCR cycle:an example
n
oC
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Thermal cycler:
The PCR machine
Agarose gel electrophoresisGel: agarose, 0.7 - 2.5 %
+
_
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Ethidium bromide
as a fluorescent stain
Agarose gel electrophoresis: analysis
Detection of DNA
fragment
determining sizes of
fragments
size
marker
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Fidelity of DNA polymerases
Optimization of PCR
Duration of annealing and elongation steps
temperature
Mg++ concentration
primer concentration template concentration
hot start (wax, Mg++ , antibodies)
touchdown PCR
additives (DMSO, betaine)
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Variations of PCR
nested PCR
inverse PCR
panhandle PCR
quantitative PCR
multiplex PCR
asymmetric PCR
in situPCR etc.
cDNA
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Isolation of mRNA by affinity chromatography
detection of DNA/RNA fragments (fordiagnostic purposes)
amplification of DNA for detection ofpolymorphism and other analyticaltechniques (sequencing, DGGE [and related
techniques], etc.)
cloning of genes (degenerate primers,differential display)
mutation of genes
gene construction
Uses of PCR and RTPCR
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PCR-based
detection of
polymorphism
Comparing genomes:The Random Amplified Polymorphic DNA
(RAPD) technique
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Comparing genomes:
The Amplified Fragment Length Polymorphism
(AFLP) technique
Comparing genomes:
The Restriction Fragment Length Polymorphism(RFLP) technique
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Detection of gene polymorphism
PCR-RFLP
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Temperature Gradient Gel Electrophoresis
(TGGE)
increasing
temperature
Variation:
Temporal Temperature Gradient Gel Electrophoresis Denaturing Gradient Gel Electrophoresis
Exonuclease may be used to digest one of the two strands;
primer must be phosphorylated
(non-denaturing gel)
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Differential Display-RTPCR (DDRT-PCR)
a technique for identifying
differentially expressed genes
Differential Display-RTPCR: principle
tissue1 tissue2 tissue3 tissue(n)
extract mRNA
RT-PCR
polyacrylamide-gelelectrophoresis
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Differential Display-RTPCR:
example
ddRT-PCR of mycelial and
budding Candida albicans
Pulsed-field gel electrophoresis
standard agarose gel electrophoresis
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Pulsed-field gel electrophoresis
orthogonal field alternation gel electrophoresis
DNA labeling
synthesis of DNA probes
detection of specific fragments
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DNA labeling
Types of labels
radioisotopes (e.g., [-32P] dNTP)
fluorescent groups (fluorophores)
enzymes
fluorescent substrates
chromogenic substrates
labeling strategies
5 or 3 labeling of fragments ( using T4 kinase or
deoxynucleotidyl terminal transferase)
incorporation of labeled nucleotides during DNA
synthesis
covalent attachment of enzymes to DNA fragments
Labeled nucleotides: examples
32P
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DNA labeling with dyes or haptens:example of a two-step strategy