CAPÍTULO 2. MARCO TEÓRICO
2.1. ESTUDIOS BÁSICOS DE INGENIERÍA
The two previously described variations in the 5' flanking region of the lactase
gene were within one of the two Alu elements in this region. The Alu element closer to
Primers were designed to specifically amplify the region comprising the reported
variations. The primers were sited in the DNA sequence flanking the Alu element due
to the repetitive occunence of Alu sequences throughout the genome. This product was
designated 5F. Single strand conformation analysis was used to analyse this product
because the known base variations did not alter restriction sites. However the length of
this product was longer than recommended in the literature for efficient detection of
variation (Hayashi, 1991) and it was therefore digested with A vail to produce two
unequal length fragments (310 and 224 base pairs - see figure 5.2.2.1). The digested
product was used directly in the analysis.
Initially both denatured and non-denatured samples were tested in order to
distinguish the double and single stranded fragments. Under the conditions used the
single stranded fragments migrated more slowly than the double stranded fragments
(Figure 5.2.2.2). It is noteworthy that the single and double stranded DNA give slightly
different colours when silver stained, the double stranded DNA being browner and the
single stranded more orange. In order to determine which single stranded bands
corresponded to those produced from each A vail digestion product (Figure 5.2.2.1.),
the digestion products were sepai ated and purified. Electrophoresis of the larger
digestion product after dénaturation produced only the lower mobility bands. The
smaller fragment was shown to be represented by the faster pair of single stranded
bands.
Initial screening of 8 unrelated individuals using four different electrophoresis
conditions, namely with and without 5% glycerol and at 4-iO°C or room temperature on a 6% aery 1 amide gel in 1 x TBE, revealed no variation under two of these conditions,
but one variant pattern was seen on the gels run without glycerol at 4°C (sample 5 in
Figure 5.2.2.2) and two different patterns were seen on gels containing glycerol run at
4°C. (samples 2 and 5 in Figure 5.2.2.2). In all the further analyses of these fragments
gels were prepared with 5% glycerol and electrophoresis was conducted in the cold room.
The two SSCA variant patterns seen were shown to be inherited in a Mendelian
fashion: for example see Figure 5.2.2.3. It is noteworthy that the two patterns were
formed by the different digestion products. Thus it can be concluded that the pattern
due to the presence of a '3' allele which occurs in the larger fragment (track 2 in figure
5.2.2.2), and is further 5' than the variation due to '2' allele in the smaller digestion
product (track 5 in Figure 5.2.2.2). The '2' allele was observed in both heterozygous
and homozygous form and comprised two bands of faster mobility than the two bands
of the more common allele (T'). On some gels the slower migrating allele 2 band and
the faster allele 1 band were indistinguishable. The '3' allele was never detected in the
homozygous forni and therefore it is not known with which band the other strand of
single stranded DNA co-migrates.
During these analyses a variation in the double stranded DNA was noted. It was
not possible to type the two types of homozygote but heterozygotes were
distinguishable owing to the presence of presumed heteroduplex bands above the
homoduplex DNA, for example see in the children in figure 5.2.2.3. This variation was
typed separately from the SSCA variation as S (slow) or F (fast). S/F occurs within the
same digestion product as '2' but was demonstrated not to be identical to '2' because
individuals heterozygous for S/F were not always heterozygous for 1/2 as detected by
SSCA.
A sample of 50 pairs of CEPH parents were analysed, and any informative
matings followed in the children and grandparents. A series of unrelated individuals
se n se
prim er A p I I
3 1 0 b ase pan s 2 2 4 base pairs
a n tisen se prim er
Figure 5.2.2.1.
Diagram o f the 5F PCR product showing the site of the A v a il recognition site and the sizes of the digestion products.
with glycerol SX I, • é sss without glycerol sss Figure 5.2.2.2.
SSCA o f A v a il digested 5F PCR product in the presence and absence of glycerol in the gel mix. The smaller digestion product is identified as S and the larger as L. The single stranded DNA produced by each fragment is identified by the prefix SS. The layout of the two gels is identical.
i ù 6ù 6 i 6
S z : = = : z s z = z : c =
It »: 23
I* E3
B
3# ## — . i - é *>« M M — ✓ y ^ î î 54 1 1 1 12 1 12 12 12 12 12 12 1 12 S S II II II II II II II II II S S Kh Figure 5.2.2.3P h o to g ra p h of a gel show ing SSCA analysis o f the A v a il dig ested 5F
PC R p ro d u c t in sam ples from C EPH fam ily 12. The track Kb corresponds to
the size markers 1 kb ladder. The lower two sets of bands conespond to the double
stranded DNA and the upper two sets to single stranded DNA. The bands with the
lowest mobility were shown to the single strands produced by the larger double stranded
component and the lower pair of single stranded by the smaller fragm ent. The variation
in the lower set of single stranded bands was typed '2' for the faster pair of bands and T'
for the slower. Vanation in double stranded DNA (highest mobility bands) typed as H
(heterozygous) for the presence of heterduplex bands and S for a single band
(homozygous).