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Chemical Synthesis of DNA (1) : Automated Multistep Process Commonly Phosphoramidite Chemistry

The document discusses several methods for synthesizing and analyzing DNA, including: 1) Automated chemical synthesis of DNA using phosphoramidite chemistry with blocking groups to control coupling reactions between nucleotides. 2) The polymerase chain reaction allows amplification of specific DNA sequences through repeated cycles of denaturation, primer annealing, and extension. 3) DNA sequencing methods like Sanger sequencing use chain-terminating dideoxynucleotides during in vitro DNA synthesis to generate fragmented copies of different lengths that can be separated and read to determine the DNA sequence.

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Harish Bg
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24 views29 pages

Chemical Synthesis of DNA (1) : Automated Multistep Process Commonly Phosphoramidite Chemistry

The document discusses several methods for synthesizing and analyzing DNA, including: 1) Automated chemical synthesis of DNA using phosphoramidite chemistry with blocking groups to control coupling reactions between nucleotides. 2) The polymerase chain reaction allows amplification of specific DNA sequences through repeated cycles of denaturation, primer annealing, and extension. 3) DNA sequencing methods like Sanger sequencing use chain-terminating dideoxynucleotides during in vitro DNA synthesis to generate fragmented copies of different lengths that can be separated and read to determine the DNA sequence.

Uploaded by

Harish Bg
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Chemical

Synthesis of
DNA (1)
• Automated multistep
process
• Commonly
phosphoramidite
chemistry
Automated DNA Synthesis (2)

• First nucleotide
attached to matrix
• DMT is a blocking
group which is
removed before the
first coupling cycle
Automated DNA Synthesis (3)

• Phosphoramidite
nucleotide
• 3’phosphite end is
the reactive portion
• DMT blocks 5’end
DNA Synthesis (4): Coupling Reaction
DNA Synthesis (5): Capping

• “Permanently
blocks unreacted
sites
• Prevents N-1
length products
DNA Synthesis (6) Oxidation

• Phosphite triester
linkage is oxidized
to pentavalent
phosphate triester
• Entire cycle can
now be repeated
Overall Yield of Chemically Synthesized
Oligonucleotides for Various Coupling
Efficiencies for Each Cycle
Nucleotide Sequences Deduced
from Amino Acid Sequences

Example A shows one possible


nucleotide sequence for a three
amino acid sequence, while
Figure B shows the effect of
degenerate sites on the possible
number of oligonucleotide
sequences to be made
Linkers and Adapters
• Linkers are generally
double stranded and
blunt ended
oligonucleotides
containing a restriction
enzyme cutting site
• Adapters generally
change a specific end
from restriction enzyme
cleavage to another
enzymes cleavage site
Cloning With Linkers

Fragments from
blunt cutting RE
Adding RE Sites Using an Adaptor

New site in vector

Sticky ends on fragment


Assembly of a Synthetic Gene
(1)

• Synthesize
overlapping
polynucleotides
which collectively
encompass entire
gene
• Ligate using T4
DNA ligase
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
DNA Sequence Determination (1)

• Fred Sanger – chain


termination method
• A normal
(2’deoxyribonucleotide)
and a 2’,3’-
dideoxyribonucleotide
(chain terminator)
DNA Sequence Determination (2)

• Normal DNA
synthesize using
2’deoxyribonucleotide
• Chain elongation still
possible on 3’end
DNA Sequence Determination (3)

• Incorporation of a
2’,3’dideoxyribonucleotide
• Further chain elongation
not possible due to lack of
free 3’OH group
Primer Extension
During
Dideoxyribonucleotide
DNA Sequencing
Reactions
1. Primer sets start point for all
fragments synthesized

2. Note that all fragments of a


given length end with the
same dideoxyribonucleotide
3. With color-coded dyes all
four reactions are possible in
one tube
DNA Sequence Determination (5):
Electrophoretic Sizing
• Fragments are sized by
electrophoretic
migration
 Gel electrophoresis
 Capillary gel
electrophoresis
• Actual sequence
shown at right
Automated DNA Sequencing

• Laser dyes
• Capillary gel
electrophoresis
• Fluorescent detection
• Example of “old”
autoradiogram with
radioactive products
shown at bottom
M13 Cloning and Sequencing Vector
• ssDNA phage with dsDNA
replication form
• Cloning into ds form
• Phage modified to have MCS
and lacZ reporter gene
• Cloning into MCS inactivates
B-galactosidase gene (no X-
gal to blue conversion
• Commonly use ss forms for
DNA sequencing templates
Cloning Into M13
Vectors (2)
• Available in
M13mp18 and M13
mp19 forms
 MCS reversed
• Allows ss templates
for sequencing both
strands of target
duplex to be
produced
Primer Walking
• Synthesize second
sequencing primer from
3’end of new sequence
determined
• Repeat as necessary to
obtain full length
sequence
• Each step “normally”
advances abut 500 or so
base pairs
• Complementary strand
should also be completed
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
Taq DNA polymerase I
– Repeat cycle 30-40 times
Polymerase
Chain Reaction
• After 1 cycle copies
double
• After 2 cycles copied
doubled and new
strand is unit length
(goes only from
primer to primer)
Polymerase
Chain Reaction

• After 3 cycles copies are


increased 8-fold and new
copies are ds and unit
length
• Odd length copies
increase arithmetically
• Unit length copies
increase in number
geometrically
Polymerase Chain Reaction
• By completion of
30-40 cycles nearly all
copies are ds DNA
and unit length
products
Gene Synthesis by PCR (1)

Used to amplify genes too large


for single step amplification

With more processive enzymes


more commonly used today to fuse
5’ and 3’ ends of related but
different genes or to carry out other
types of genetic engineering
Gene Synthesis by PCR (2)
PCR-Based DNA Sequencing
Requires much less
template

Uses a single primer


and dideoxynucleotides

Most common method


used today

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