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KLAUS DEICHMANN
• Stop Solution
The stop solution inactivates the enzyme and denatures the hybrids which
have formed.
• Polyacrylamide Gels
Good qualityis essential for sharp bands and a uniform pattern. Always use
fresh acrylamide/bisacrylamide solution and electrophoresis grade materials. Long range sequencing can be achieved by long gels ( 80 to 100 cm),
gradient gels differing in acrylamide concentration from top to bottom, or
by using wedged spacers.
Mechanism of dideoxy chain termination sequencing
The sequencing protocol using the chain termination method as described
by Sanger differs somewhat according to the polymerase used. The following refers to the use of modified T7 DNA polymerase. After preparation of
the recombinant vectors, sequencing is carried out in 4 steps.
1. First annealing has to be performed using approximately equimolar
amounts of template DNA and primer. Heating to 65°C prevents formation of secondary structures of the template DNA. Cooling slowly over
several minutes allows annealing of the primer.
2. For the labeling reaction, equimolar amounts of dNTPs are added, one of
which is radio-labeled (usually a[ 35 S]dATP). The polymerase is added
and polymerization performed for a few minutes (this step must be performed under the right buffer conditions). The result is radioactively labeled complementary strands of heterogenaus size ranging from a few
base pairs to about 200 bp.
3. For the termination reaction the labeled mixture is divided into 4 tubes
and equimolar amounts of all 4 dNTPs are added as well as a small
amount of ddNTP (a different one in each of the 4 tubes). The enzyme
will still be active. Incubation is carried out for several minutes, and
chain termination occurs in each position where a dideoxy nucleotide
is incorporated (see Figure 3).
4. By adding the stop solution to the reactions, the enzyme is inactivated
and the template-terminated chain hybrids are denatured, prior to loading them on a sequencing gel ( usually a denaturing polyacrylamide gel).
KLAUS DEICHMANN
• Stop Solution
The stop solution inactivates the enzyme and denatures the hybrids which
have formed.
• Polyacrylamide Gels
Good qualityis essential for sharp bands and a uniform pattern. Always use
fresh acrylamide/bisacrylamide solution and electrophoresis grade materials. Long range sequencing can be achieved by long gels ( 80 to 100 cm),
gradient gels differing in acrylamide concentration from top to bottom, or
by using wedged spacers.
Mechanism of dideoxy chain termination sequencing
The sequencing protocol using the chain termination method as described
by Sanger differs somewhat according to the polymerase used. The following refers to the use of modified T7 DNA polymerase. After preparation of
the recombinant vectors, sequencing is carried out in 4 steps.
1. First annealing has to be performed using approximately equimolar
amounts of template DNA and primer. Heating to 65°C prevents formation of secondary structures of the template DNA. Cooling slowly over
several minutes allows annealing of the primer.
2. For the labeling reaction, equimolar amounts of dNTPs are added, one of
which is radio-labeled (usually a[ 35 S]dATP). The polymerase is added
and polymerization performed for a few minutes (this step must be performed under the right buffer conditions). The result is radioactively labeled complementary strands of heterogenaus size ranging from a few
base pairs to about 200 bp.
3. For the termination reaction the labeled mixture is divided into 4 tubes
and equimolar amounts of all 4 dNTPs are added as well as a small
amount of ddNTP (a different one in each of the 4 tubes). The enzyme
will still be active. Incubation is carried out for several minutes, and
chain termination occurs in each position where a dideoxy nucleotide
is incorporated (see Figure 3).
4. By adding the stop solution to the reactions, the enzyme is inactivated
and the template-terminated chain hybrids are denatured, prior to loading them on a sequencing gel ( usually a denaturing polyacrylamide gel).