CAPILLARY ELECTROPHORESIS DNA SEQUENCING
Content
Introduction .............................................................................................................................................2
Materials for DNA Sequencing ...............................................................................................................3
Method for DNA Sequencing............................................................................................................... 4-9
Prepare Genomic DNA Template ............................................................................................................4
PCR amplification ............................................................................................................................... 4-6
Determine DNA quantity .................................................................................................................... 6-7
Sequencing reaction preparation ......................................................................................................... 7-8
Purification of extension product ........................................................................................................ 8-9
Perform Capillary Electrophoresis ...........................................................................................................9
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CAPILLARY ELECTROPHORESIS DNA SEQUENCING
Introduction
Sanger sequencing is a DNA sequencing method in which target DNA is denatured and annealed to
an oligonucleotide primer, which is then extended by DNA polymerase using a mixture of
deoxynucleotide
triphosphates
(normal
dNTPs)
and
chain-terminating
dideoxynucleotide
triphosphates (ddNTPs). ddNTPs lack the 3’ OH group to which the next dNTP of the growing DNA
chain is added. Without the 3’ OH, no more nucleotides can be added, and DNA polymerase falls off.
The resulting newly synthesized DNA chains will be a mixture of lengths, depending on how long the
chain was when a ddNTP was randomly incorporated.
Most DNA sequencing is now automated. The Sanger method chain termination reactions are still
used, but pouring, running, & reading polyacrylamide gels has been replaced by automated methods.
Instead of labeling the products of all 4 sequencing reactions the same (with a radioactive
deoxynucleotide), each dideoxynucleotide is labeled with a different fluorescent marker. When excited
with a laser, the 4 different kinds of products are detected and the fluorescence intensity translated into
a data “peak”. Thus all four chain termination reactions can be performed in the same tube, and run on
a single lane on a gel. A machine scans the lane with a laser. The wavelength of fluorescence from the
label conjugated to the ddNTPs can be interpreted by the machine as an indication of which reaction
(ddG, ddA, ddT, or ddC) a particular DNA band came from.
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CAPILLARY ELECTROPHORESIS DNA SEQUENCING
Materials for DNA Sequencing

Template DNA

dNTP mix

Distilled water

Enhancing buffer

Primers
o Forward
o Reverse
 PCR mix

Taq DNA polymerase

EXO-SAP enzyme

Purification column

Dye terminator mix

Sephadex

Running buffer

Sequence polymer

Sequence plate

Plate septa
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CAPILLARY ELECTROPHORESIS DNA SEQUENCING
Method for DNA Sequencing
A. Prepare Genomic DNA Template
a. Genomic DNA is prepared according to Standard protocols for isolating high-molecular
weight DNA.
b. Genomic DNA concentration is determined by A260, dye incorporation or an equivalent
spectrophotometric analysis.
c. According to determined concentration, genomic DNA is diluted with sterile, deionized
water or 10 mM Tris, pH 8.0 in order to obtain DNA concentration in between 30-60
ng/μL.
d. After utilization, diluted DNA is aliquoted and stored at -15°C to -25°C.
e. DNA quality is determined by calculation of A260/A280 ratio.
Note: For high quality DNA, the ratio A260/A280 is required to be in between 1.7 to 1.9
B. PCR amplification
Preparation of PCR reaction:
i. Reaction mix is prepared.
ii. If number of samples to be analyzed is more than 5, it is recommended to prepare
master mix. Depending on the calculated volume, combine the components and
vortex gently.
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CAPILLARY ELECTROPHORESIS DNA SEQUENCING
Reagents to be used:
x…………..…………….. 12,5 μl
PCR master mix includes chemicals required for polymerase chain reaction as
MgCl2, dNTP, Taq polymerase and buffer.
Forward Primer to be used in reaction has to be designed appropriate for
genomic DNA region of interest.
Reverse Primer to be used in reaction has to be designed appropriate for
genomic DNA region of interest.
It catalyzes binding of primers to DNA region of interest.
-60 μl)……………. 2,5 μl
After all reactions are added, vortex the pcr tube gently in order to obtain properly mixed
chemicals. Total volume is 25 μl.
conditions of activation,
amplification and extention according to types of used chemicals and genomic
DNA region of interest.
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CAPILLARY ELECTROPHORESIS DNA SEQUENCING
An illustrative pcr protocol is following:
Activation: No repeat
95°C………………… 10’
Amplification: 40 repeat
95°C………………… 40’’
62°C………………… 1’
72°C………………… 50’’
Elongation: No repeat
72°C………………… 7’
Hold:
4°C………………… ∞
C. Determine DNA quantity
a. In order to determine the quantity of PCR product mix 5 μl of PCR product with loading
dye.
b. Prepare 2% agarose jel and perform electrophoresis.
c. Load low mass DNA marker into one well of agarose gel.
d. Visualise the gel under UV illuminator and analyze the image acquired.
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CAPILLARY ELECTROPHORESIS DNA SEQUENCING
e. Clean up protocol for PCR product
f.
Briefly centrifuge the pcr tubes before usage.
g. Label new pcr tubes according to sample names.
h. Add 5 μl of pcr product.
i.
Add 2 μl of specific type of enzyme to clean up the un-utilized pcr components in the
tube.
j.
Total volume is 7 μl.
k. Place the tubes into Thermal Cycler.
l.
Set up the conditions depending on the enzyme used to clean excessive reagents up.
An illustrative clean up protocol is following:
Enzyme incubation: 37°C………………… 30’
Enzyme inactivation: 80°C………………… 15’
Hold: 4°C………………… ∞
D. Sequencing reaction preparation
Components of the reaction and amount of chemicals to be used:
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CAPILLARY ELECTROPHORESIS DNA SEQUENCING
Total volume is 10 μL. Vortex the tubes to mix components properly and spin down briefly.
Place the tubes into Thermal Cycle and set the conditions.
An illustrative pcr condition is following:
Activation: No repeat
96°C………………… 1’
Extention: 25 cycle
96°C………………… 10’’
50°C………………… 5’’
60°C………………… 4’
Hold:
4°C………….……… ∞
E. Purification of extension product
Extended products are purified by using Sephadex colons.
Preparation of purification columns:
a. Add 750 μL of ddH2O to columns including sephadex
b. Vortex the colons till the Sephadex powder is dissolved properly and keep at RT for 30 min.
c. Centrifuge the columns for 2 minutes at 4600 rpm
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CAPILLARY ELECTROPHORESIS DNA SEQUENCING
d. Discard the water in the collecting tube and assure that there is no water left.
e. Add whole PCR product to the center of column.
f. Centrifuge for 2 minutes at 4600 rpm.
F. Perform Capillary Electrophoresis
a. Add all the purified product into 96 well plate.
b. Cover the plate with Septa.
c. Place the plate into plate holder.
d. Load the plate into DNA sequencing instrument.
e. Wait instrument to perform electrophoresis and collect data
f. Analyze the data.
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