Instruction manual

Section 4
Sample and Reagent Preparation
4.1 Sample Preparation
In some cases, adding a high melting domain to the DNA fragment allows one to analyze
mutations that normally are not seen.
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The addition of a 30–40 base pair GC clamp to the
DNA fragment during PCR creates a high melting domain and will influence the other melt-
ing domains.
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As a result, the sequence of interest should be in the first (low) melting domain
and therefore, can be analyzed. A GC clamp is not needed if the sequence of interest resides
in a low melting domain and has one end of the fragment containing a natural high melting
domain. The use of melting profile programs, such as MacMelt software, can show regions
of theoretical high and low melting domains of a known sequence. These programs can help
determine if a GC clamp will allow better detection of mutations.
If GC clamps are not used, this might cause missing of the transition zone and show a
strong single strand band in a perpendicular DGGE gel. In this case, it might be a good idea
to add a GC clamp to one end of the fragment.
As mentioned above, the use of GC clamps helps in the ability to detect mutations. An
alternative to GC clamps is using psoralen derivative PCR primers called ChemiClamp
primers.
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Psoralens are photoreagents that form covalent bonds with pyrimidine bases of
nucleic acids. Psoralen-oligonucleotide conjugates allow crosslinking of DNA fragments at
one end by photoinduction with a UV source. Because ChemiClamps covalently link the two
DNA strands at one end, they should not be used when isolating a DNA fragment which is
going to be sequenced from a gel.
Samples run in the DGGE gel are typically prepared by PCR. The PCR samples can be
loaded onto the gel after PCR without any other manipulations. The size of the DNA fragments
run on DGGE should be in the 100–800 bp range, although DNA fragments as long as 1,000 bp
can also be analyzed.
4.2 Reagent Preparation
The concentration of denaturants varies for samples analyzed in the D GENE system.
The concentration of acrylamide can vary as well, depending on the size of the fragment that
is being analyzed. Both 0% and 100% denaturants should be made as stock solutions. A 100%
denaturant is a mixture of 40% deionized formamide and 7 M urea. The following reagents
are included in the D GENE Electrophoresis Reagent Kit, catalog number 170-9032.
40% Acrylamide/Bis (37.5:1)*
Acrylamide 38.0 g
Bis-acrylamide 2.0 g
Add dH
2
O to 100 ml. Filter through a Whatman No. 1 and store at 4 °C.
* Polyacrylamide gels are described by reference to two characteristics:
1) The total monomer concentration (%T)
2) The crosslinking monomer concentration (%C)
%T =
gm acrylamide + gm Bis-acrylamide
x 100
Total Volume
%C =
gm Bis-acrylamide
x 100
gm acrylamide + gm Bis-acrylamide
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