Two different techniques were used for analyzing the restriction fragment length polymorphisms. As the length of the analyzed fragments varied between 125 and 919 base pairs, agarose gels turned out to be a difficult means for resolving all fragments. For different fragments, different agarose concentrations were used (1.5 to 3%), but the differentiation between the uncut fragment II (919 bp) and cut fragment (717 bp) remained difficult. The differentiation between cut and uncut fragment III (24 base pairs) was resolvable by using high concentration agarose gels (3%). Anyhow, the yield of a 200 base pair fragment during ethanol precipitation is low (see section EtOH), so the amplification volume for PCR had to be doubled. Another problem arising during establishing the protocol was the loss of evidence after photographing the agarose gels.
DNA separations are most frequently performed in large sequencing polyacrylamide gels or in agarose gels in presence of the fluorescent dye ethidium bromide (see section EtBr). The agarose method exhibits a low resolution, relatively low sensitivity and requires a photograph for a storable pherogram. The method employing long sequences gels has the disadvantage of radioactive labelling and is time consuming. Because of these drawbacks, another protocol was introduced for clinical applications.
Thin, washed, and dried polyacrylamide gels with sample wells (CleanGels, Pharmacia), bound to a polyester backing film, which were rehydrated to 0.5 mm thick gels with a specially designed buffer, were used. They were applied together with a highly sensitive silver staining (see section silver). These pherograms can be easily dried for evaluation and filing of the results.
The discontinuous polyacrylamide gels
(stacking gel T=5%, C=3% and separating gel T=10%, C=2%) were
rehydrated with 25 ml of a 112 mM Tris-acetate buffer (acetic acid to pH 6.4),
containing 0.001% bromophenol blue and 0.001% Orange G, placed on a rotating
shaker for 60 minutes to assure even rehydration. The excess buffer was removed
with clean filter paper, sample wells dried and the buffer wiped of the gel
surface. Electrophoresis was performed using a Multiphor II Electrophoresis
Unit (Pharmacia), connected to a MultiTemp Thermostatic Circulator (Pharmacia),
set to a temperature of 15psy176 C. A very thin layer of kerosene was applied
onto the cooling plate in order to ensure good cooling contact. The gel was
placed on to the center of the cooling plate, the side containing the wells
oriented towards the cathode. Two electrode wicks were soaked with 22 ml of an
electrode buffer (0.20 mM Tris, 0.2 mM Tricin, 0.55% SDS, pH 8.3 with acetic
acid [141]) each. One electrode strip was placed onto the edge of
the gel so that there was a distance of 4 mm between the edge of the strip and
the sample wells. Another strip was placed onto the other edge of the gel so
that the strip overlapped the gel by 5 mm. Air bubbles were smoothed out by
sliding bent-tipped forceps along the edges of the strips lying in contact with
the gel. 7 
l sample per well were applied. No sample preparation was
performed apart from ethanol precipitation. The running conditions for a whole
gel are given below.
Table 4.6: Running conditions for CleanGels
Electrophoresis was performed until the bromophenol blue front reached the anode.