13 Sequencing
193
Automated fluorescent sequencing
This method is based on the use of dye-labeled dideoxynucleotides as terminators of the DNA elongation (Prober et al. 1987; Wilson et al. 1988).
Replacing normal ddNTPs and radioactive labeled dNTPs with the dye-labeled terminators provides 3 advantages: First, radioactive work can be
avoided, facilitating sequencing. Second, all four termination reactions
can be performed in a single tube provided that four different dyes are
used, one for each of the ddNTPs. The method reduces tube handling
and pipetting by a factor of four. Third, using different dyes, all reactions
may be loaded into one lane for gel electrophoresis. The bands are then
recognized by their ability to absorb light of different wavelengths when
passing a scanner. Modern sequencing machines combine the advantages
ofTaq polymerase sequencing and the use of dye-labeled ddNTPs- the basis
for modern automated sequencing.
The protocol given below describes reactions using the same dye. The
reaction products are suited for use in many of the modern sequencing machines.
Multiplexing
Multiplexing represents a method to speed up sequencing by running several different sequencing reactions in the same 4 lanes of a gel at one time
(Church and Kieffer-Higgins 1988). The different reactions arenot radiolabeled, but initiated by the use of different primers. The sequencing gel is
blotted onto a membrane after electrophoresis has been performed, and
the blot is hybridized with a radioactive probe complementary to one of
the primers, thereby giving signals in all four lanes corresponding to
one template. After reading the film, the probe is washed off and hybridization performed using the next primer-probe. Multiplexing is a useful tool to
save time and material when sequencing large amounts of DNA (e.g. the
Human Genome Project).
Labaratory protocols
The following two sequencing protocols will provide standard reaction conditions usingmodified T7 DNA polymerase (Sequenase™) orTaqpolymerase. They should be optimized for a particular application. Additional information may be found in the troubleshooting section. Most commercial
193
Automated fluorescent sequencing
This method is based on the use of dye-labeled dideoxynucleotides as terminators of the DNA elongation (Prober et al. 1987; Wilson et al. 1988).
Replacing normal ddNTPs and radioactive labeled dNTPs with the dye-labeled terminators provides 3 advantages: First, radioactive work can be
avoided, facilitating sequencing. Second, all four termination reactions
can be performed in a single tube provided that four different dyes are
used, one for each of the ddNTPs. The method reduces tube handling
and pipetting by a factor of four. Third, using different dyes, all reactions
may be loaded into one lane for gel electrophoresis. The bands are then
recognized by their ability to absorb light of different wavelengths when
passing a scanner. Modern sequencing machines combine the advantages
ofTaq polymerase sequencing and the use of dye-labeled ddNTPs- the basis
for modern automated sequencing.
The protocol given below describes reactions using the same dye. The
reaction products are suited for use in many of the modern sequencing machines.
Multiplexing
Multiplexing represents a method to speed up sequencing by running several different sequencing reactions in the same 4 lanes of a gel at one time
(Church and Kieffer-Higgins 1988). The different reactions arenot radiolabeled, but initiated by the use of different primers. The sequencing gel is
blotted onto a membrane after electrophoresis has been performed, and
the blot is hybridized with a radioactive probe complementary to one of
the primers, thereby giving signals in all four lanes corresponding to
one template. After reading the film, the probe is washed off and hybridization performed using the next primer-probe. Multiplexing is a useful tool to
save time and material when sequencing large amounts of DNA (e.g. the
Human Genome Project).
Labaratory protocols
The following two sequencing protocols will provide standard reaction conditions usingmodified T7 DNA polymerase (Sequenase™) orTaqpolymerase. They should be optimized for a particular application. Additional information may be found in the troubleshooting section. Most commercial