C.B.S. Scientific DTSK-2401-220 - Manual (Page 5)

Cipher Genetic Analysis System
DTSK Instruction Manual, version 8/30/2011
5
www.cbsscientific.com
SECTION 1
General Information
1.1 Introduction
DESCRIPTION
The C
ipher
G
enetiC
A
nAlysis
s
ystems
(DTSK-2001 and DTSK-2401) allow the researcher to choose which technique
is most applicable to their investigation. Both DGGE and TTGE rely on establishing a gradient of either solvent
(Urea/Formamide) or temperature in which the target fragments will undergo conformational transition (melt). This
sequence dependent information will determine the theoretical melting behavior of the target fragment after PCR
amplification. If the sequence is known, then primer/probe design can be made using certain software design
programs (1). If not, the melting range can be revealed by running perpendicular DGGE/TTGE gels. The third
mutation detection technique, SSCP, differentiates between normal and mutant duplex DNA by denaturing the DNA
to form single stranded molecules of equal length. These molecules can re-anneal onto themselves and based on
the varying degree of intrastrand base pairing, form different three-dimensional structures. These structures, differ
in electrophoretic mobility and can be separated on a non-denaturing polyacrylamide gel.
C.B.S. SCIENTIFIC has designed two different C
ipher
G
enetiC
A
nAlysis
s
ystems
which are reliable and easy-to-use.
The DTSK-2001 is a 2 gel system and includes two single gel cassettes. The DTSK-2401 is a four gel system which
includes two dual gel cassettes. The systems feature: programmable heater/stirrer which can be programmed for
DGGE, TTGE and SSCP applications, a large diameter cooling coil for use with an external chiller, a simplified
method for casting perpendicular and vertical gels using Gel Wrap ® , single or dual gel cassettes, an internal
impellor pump for buffer cycling, polypropylene spring clamps, and a safety cover with an electrical interlock which
helps maintain temperature, reduce evaporation, and protect against shock hazard.
DGGE & CDGE
Denaturing Gradient Gel Electrophoresis (DGGE) is a powerful genetic analysis technique that can be used for
detecting single base changes and polymorphisms in genomic (2,3), cloned, and PCR amplified DNA (3,4). Two
of the most valuable uses for DGGE in human, animal or microbial genetics are in directly detecting single base
changes that cause disease and in detecting polymorphisms with DNA probes for genetic-linkage analysis. In DGGE,
conformational transitions of multiple nucleic acid complexes are induced by an increasing concentration of solvent
(Urea/Formamide) at a constant temperature. Clinical applications of DGGE include a rapid and effective method
for screening samples for genetic mutations and variants. Also, DNA fragment melting points can be determined
using perpendicular DGGE (2). In contrast to DGGE, CDGE (Constant Denaturant Gel Electrophoresis) uses a
single solvent percentage to induce partial melting of DNA fragments as they enter the gel. The disadvantage of
CDGE is that only a single melting domain can be interrogated.
TTGE
Destabilization of nucleic acid complexes can also be studied using acrylamide gels which contain a uniform solvent
concentration (Urea/Formamide), but with an increasing temperature gradient (6). Since the temperature of the
entire gel is uniformly raised over a period of time, this technique has been termed ‘TTGE’, or Temporal Temperature
Gradient Electrophoresis (7). This technique incorporates many improvements over DGGE/CDGE especially when
studying multiple melting domains (8).
SSCP
Single Strand Conformational Polymorphism (SSCP) reveals differences in electrophoretic mobility between normal
and mutant single strands of DNA (9). In SSCP, normal and mutant duplex DNA are denatured to form single stranded
molecules of equal length. These molecules can re-anneal onto themselves and based on the varying degree of
intrastrand base pairing, form different three-dimensional structures. These structures, differ in electrophoretic
mobility and can be separated on a chilled non-denaturing polyacrylamide gel. The electrophoretic mobility of
these “conformers” change depending on temperature and buffer ionic strength. For accurate characterization
of mutations within these re-folded single strands, it is essential that the buffer temperature be tightly controlled
within each electrophoresis procedure, usually between 4º-25ºC. If the gel temperature is not precisely controlled,
the resolution will suffer because of the loss of intrastrand base pairing and change in the overall shape of the
3-dimensional conformer.