Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 49)

A variation of heteroduplex analysis is Conformation Sensitive Gel Electrophoresis
(CSGE). This technique exploits the observation that a mildly denaturing environment will
enhance the ability of single-based mismatches to produce conformational changes.
19 These
changes also increase the differential migration of heteroduplex and homoduplex molecules.
Samples are electrophoresed in 6–10% polyacrylamide gels (99:1), tris-taurine buffer and
10% ethylene glycol with 15% formamide as denaturants. Bis (acryloyl) piperazine (BAP) or
piperazine diacrylamide (PDA) can also be used instead of bis as a cross-linker.
20 BAP or
PDA cross-linker helps to improve the gel strength and increase the pore size in the gel. PCR
fragment sizes for CSGE typically run between 300–800 bp in length for optimum mutation
detection.
21
5.2 Reagent Preparation
Heteroduplex Analysis
The concentration and type of acrylamide to use varies for the sample being analyzed on
the DCode system. Therefore, a 40% stock solution containing acrylamide and bis-acrylamide
(bis) should be made, or a 2x DEM solution. Reagents for casting and running a heteroduplex
analysis gel are included in the DCode electrophoresis reagent kit for Heteroduplex Analysis,
catalog number 170-9173.
For a different percent crosslinking, use the equation below to determine the amount of bis
to add. The example stock solution below is for an acrylamide/bis ratio of 37.5:1.
40% Acrylamide/Bis (37.5:1)
Reagent
Amount
Acrylamide 38.93 g
Bis-acrylamide 1.07 g
dH
2
O to 100.0 ml
Filter through a 0.45 µ filter 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 =
g acrylamide + g bis-acrylamide
x 100
Total Volume
%C =
g bis-acrylamide
x 100
g acrylamide + g bis-acrylamide
10x TBE Buffer
Reagent
Amount Final Concentration
Tris base 108 g 0.89 M
Boric acid 55 g 0.89 M
0.5 M EDTA, pH 8.0 40 ml 20 mM
dH
2
O to 1L
Mix and add dH
2
O to 1 L. Autoclave for 20–30 minutes. Store at room temperature.
45