84
FENG QIAN AND GREGORY G. GERMINO
This is best achieved by making a mastermix of all reagents and adding an
appropriate amount of the radionucleotide. It is not necessary to reduce the
concentration of cold dCTP in the reaction mix. After PCR amplification,
the labeled PCR product is mixed with 5 times the volume of formamide
loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue and
0.05% xylene cyanol). The samples are denatured for 5 min at 95°C and
placed directly on ice. 2 J..tl of each sample are loaded onto gels.
Electrophoresis
The electrophoresis in SSCP analysis is routinely carried out under two conditions. The first gel consists of 6% acrylamide (49:1 acrylamide:bis), 10%
glycerol and 0.5 x TBE. Sampies on this gel are electrophoresed for at least
2.5 hat 0.5 watt/cm) at room temperaturein 0.5 x TBE running buffer. Constant temperature can be maintained using a cooling fan. The second gel
condition has the same gel composition except it contains no glycerol and
electrophoresis is performed at 4°C. Gels are then transferred to 3MM
Whatman paper, dried and autoradiographed. The Ionger length of a sequencing gel apparatus (30- 50 cm) offers the advantage of Ionger run
time and less chance of running abberant bands off the bottom. W e
have had, however, excellent results using a shorter vertical gel system
such as the Protean Ilxi by BioRad. A major advantage of this system is
its internal cooling chamber. Attached to a temperature-controlled circulator, gel temperature can be easily controlled to optimize band separation
on the bench top without the need for a fan or a cold room.
Comments
Parameters affecting SSCP sensitivity
To maximize the detection of singlebasepair changes by SSCP, one should
take the following factors into account.
• Size of DNA fragments
There is a striking relationship between sensitivity and fragment length.
The optimal size for relatively sensitive detection of single base changes
is approximately 200 bp or less. For larger fragments, this Iimitation can
be overcome by digesting the DNA with appropriate restriction endonucleases to reduce the fragment sizes prior to SSCP assay.
FENG QIAN AND GREGORY G. GERMINO
This is best achieved by making a mastermix of all reagents and adding an
appropriate amount of the radionucleotide. It is not necessary to reduce the
concentration of cold dCTP in the reaction mix. After PCR amplification,
the labeled PCR product is mixed with 5 times the volume of formamide
loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue and
0.05% xylene cyanol). The samples are denatured for 5 min at 95°C and
placed directly on ice. 2 J..tl of each sample are loaded onto gels.
Electrophoresis
The electrophoresis in SSCP analysis is routinely carried out under two conditions. The first gel consists of 6% acrylamide (49:1 acrylamide:bis), 10%
glycerol and 0.5 x TBE. Sampies on this gel are electrophoresed for at least
2.5 hat 0.5 watt/cm) at room temperaturein 0.5 x TBE running buffer. Constant temperature can be maintained using a cooling fan. The second gel
condition has the same gel composition except it contains no glycerol and
electrophoresis is performed at 4°C. Gels are then transferred to 3MM
Whatman paper, dried and autoradiographed. The Ionger length of a sequencing gel apparatus (30- 50 cm) offers the advantage of Ionger run
time and less chance of running abberant bands off the bottom. W e
have had, however, excellent results using a shorter vertical gel system
such as the Protean Ilxi by BioRad. A major advantage of this system is
its internal cooling chamber. Attached to a temperature-controlled circulator, gel temperature can be easily controlled to optimize band separation
on the bench top without the need for a fan or a cold room.
Comments
Parameters affecting SSCP sensitivity
To maximize the detection of singlebasepair changes by SSCP, one should
take the following factors into account.
• Size of DNA fragments
There is a striking relationship between sensitivity and fragment length.
The optimal size for relatively sensitive detection of single base changes
is approximately 200 bp or less. For larger fragments, this Iimitation can
be overcome by digesting the DNA with appropriate restriction endonucleases to reduce the fragment sizes prior to SSCP assay.