Appeal No. 95-3606
Application 07/827,691
PCR the 201 and 214 primers (Fig. 2) were utilized. The
products of this reaction were used as template for the
second-stage 203- and 213-primed PCR. The products of
these reactions were then subcloned into M13 for sequence
analysis. This analysis revealed that only the length of
the repeat sequences varied--the flanking sequences
between
the PCR primers and the repeat remaining the same (Fig.
1B).
In all cases the cloned PCR products were substantially
shorter than anticipated, particularly since the fragile
X
individuals had large insertions or amplifications of
sequences in this region . . . .
Fig. 3 is a “Southern blot analysis with fragile X-
affected and normal males” (Kremer, p. 1713, Fig. 3):
Total genomic DNA from lymphocytes was extracted and
purified. A portion of each sample . . . was digested
to completion with (A)Pvu II and Bam HI, (B) Pvu II and
Nhe I, (C) Sau 3AI, (D) PST I and Rsa I. Lanes 1, 2,
and 5 are from affected males. Lanes 3 and 4 are from
normal males. Samples were separated by electrophoresis
on a 1.3% agarose gel and transferred to Hybond N+
blotting
membrane (Amersham). The probe pfxa4 was P-labeled by32
random priming and hybridized to the blots . . . and
exposed to Xomat XK-1 film . . . .
On analysis, Kremer states (Kremer, p. 1713, col. 2, to
p.1714, col. 1; emphasis added):
We have concluded from all the experimental evidence
that the unstable DNA sequence which characterizes the
fragile X genotype maps to the p(CCG) trinucleotide
n
repeat.
We have demonstrated that normal X chromosomes have about
40 + 25 copies of p(CCG) and that within these limits
n
the sequence is a stable DNA polymorphism. The fragile X
- 12 -
Page: Previous 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Next
Last modified: November 3, 2007