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8/9/2019 Biotech Lect Ch05 Mod
http://slidepdf.com/reader/full/biotech-lect-ch05-mod 1/29
Chemical
Synthesis of DNA (1)
Automated multistep
process Commonly
phosphoramidite
chemistry
8/9/2019 Biotech Lect Ch05 Mod
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Automated DNA Synthesis (2)
First nucleotide
attached to matrix
DMT is a blockinggroup which is
removed before the
first coupling cycle
8/9/2019 Biotech Lect Ch05 Mod
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Automated DNA Synthesis (3)
Phosphoramidite
nucleotide
3¶phosphite end isthe reactive portion
DMT blocks 5¶end
8/9/2019 Biotech Lect Ch05 Mod
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DNA Synthesis (4): Coupling Reaction
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DNA Synthesis (5): Capping
³Permanently
blocks unreacted
sites Prevents N-1
length products
8/9/2019 Biotech Lect Ch05 Mod
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DNA Synthesis (6) Oxidation
Phosphite triester
linkage is oxidized
to pentavalent phosphate triester
Entire cycle can
now be repeated
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Overall Yield of Chemically Synthesized
Oligonucleotides for Various CouplingEfficiencies for Each Cycle
8/9/2019 Biotech Lect Ch05 Mod
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Nucleotide Sequences Deduced
from Amino Acid Sequences
Example A shows one possible
nucleotide sequence for a three
amino acid sequence, whileFigure B shows the effect of
degenerate sites on the possible
number of oligonucleotide
sequences to be made
8/9/2019 Biotech Lect Ch05 Mod
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Linkers and Adapters
Linkers are generally
double stranded and
blunt ended
oligonucleotidescontaining a restriction
enzyme cutting site
Adapters generally
change a specific endfrom restriction enzyme
cleavage to another
enzymes cleavage site
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Cloning With Linkers
Fragments from
blunt cutting RE
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Adding RE Sites Using an Adaptor
New site in vector
Sticky ends on fragment
8/9/2019 Biotech Lect Ch05 Mod
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Assembly of a Synthetic Gene (1)
Synthesize
overlapping
polynucleotideswhich collectively
encompass entire
gene
Ligate using T4DNA ligase
8/9/2019 Biotech Lect Ch05 Mod
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Assembly of a
Synthetic Gene (1)
Synthesize overlapping
polynucleotides which
leave ss gaps in gene
Fill gaps using DNAP I
Ligate with T4 DNA
ligase
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DNA Sequence Determination (1)
Fred Sanger ± chaintermination method
A normal
(2¶deoxyribonucleotide)
and a 2¶,3¶-dideoxyribonucleotide
(chain terminator)
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DNA Sequence Determination (2)
Normal DNA
synthesize using
2¶deoxyribonucleotide Chain elongation still
possible on 3¶end
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DNA Sequence Determination (3)
Incorporation of a
2¶,3¶dideoxyribonucleotide Further chain elongation
not possible due to lack of
free 3¶OH group
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Primer Extension
DuringDideoxyribonucleotide
DNA Sequencing
Reactions
2. Note that all fragments of a
given length end with thesame dideoxyribonucleotide
1. Primer sets start point for all
fragments synthesized
3. With color-coded dyes all
four reactions are possible in
one tube
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DNA Sequence Determination (5):
Electrophoretic Sizing Fragments are sized
by electrophoretic
migration Gel electrophoresis
Capillary gel
electrophoresis
Actual sequenceshown at right
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Automated DNA Sequencing
Laser dyes
Capillary gel
electrophoresis Fluorescent detection
Example of ³old´
autoradiogram with
radioactive productsshown at bottom
8/9/2019 Biotech Lect Ch05 Mod
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M13 Cloning and Sequencing Vector
ssDNA phage with dsDNA
replication form
Cloning into ds form
Phage modified to have MCSand lacZ reporter gene
Cloning into MCS inactivates
B-galactosidase gene (no X-
gal to blue conversion
Commonly use ss forms for
DNA sequencing templates
8/9/2019 Biotech Lect Ch05 Mod
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Cloning Into M13
Vectors (2) Available in
M13mp18 and M13
mp19 forms MCS reversed
Allows ss templates
for sequencing both
strands of targetduplex to be
produced
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Primer Walking
Synthesize secondsequencing primer from3¶end of new sequencedetermined
Repeat as necessary toobtain full lengthsequence
Each step ³normally´
advances abut 500 or so base pairs
Complementary strandshould also be completed
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Polymerase
Chain Reaction
(PCR) Allows 108+fold
amplification of specific
DNA sequences Procedure
± Denature DNA
± Anneal primers flanking
region to be amplified
± Extend primers using
T aq DNA polymerase I
± Repeat cycle 30-40 times
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Polymerase
Chain Reaction After 1 cycle copies
double
After 2 cycles copieddoubled and new
strand is unit length
(goes only from
primer to primer)
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Polymerase
Chain Reaction
After 3 cycles copies are
increased 8-fold and new
copies are ds and unitlength
Odd length copies
increase arithmetically
Unit length copies
increase in number
geometrically
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Polymerase Chain Reaction By completion of
30-40 cycles nearly all
copies are ds DNAand unit length
products
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Gene Synthesis by PCR (1)
Used to amplify genes too largefor single step amplification
With more processive enzymes
more commonly used today to fuse
5¶ and 3¶ ends of related butdifferent genes or to carry out other
types of genetic engineering
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Gene Synthesis by PCR (2)
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