Appeal No. 95-3606
Application 07/827,691
fragment was isolated from pfxa1 and subcloned into the
Pst I site in M13 mp18. . . . The 530-bp Nhe I to Pst I
restriction endonucleases fragment was also isolated from
pfxa1 and subcloned in both orientations into Xba 1-Pst I
sites in M13 mp18 and 19. The difficulties in isolating
M13 clones that spanned the p(CCG) repeat in the reverse
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direction led us to use double-stranded sequencing of
pfxa2
using oligodeoxyribonucleotide primers #201, 203, 204,
209,
and 213. All sequencing was performed with Sanger’s
dideoxy
method and with TAQuence sequencing kit (U.S. Biochemical
Corp.). Because of high CG content of the template DNA,
samples were routinely prepared with 7-deaza-dGTP,
denatured
in a final concentration of 50% formamide at 90 C for 5O
min.
and loaded onto sequencing gels immediately without
allowing
to cool.
Under Fig. 1(B), Kremer teaches “PCR products spanning the
p(CCG) repeat were generated as described in the text and
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separated on a 1.5% low melting point argose (IBI) gel”
(Kremer, p. 1712, Fig. 1(B)). Kremer describes PCR product
generation in the text as follows (Kremer, pp. 1711-1712,
bridging para.):
In attempts to obtain sequence data for this region
from normal individuals and additional fragile X genotype
individuals, two approaches were undertaken. The first
used two-stage PCR. Starting material was either total
chromosomal DNA or, in one case, Eco RI-digested DNA from
a normal individual; the DNA was fractionated by agarose
gel electrophoresis to enrich for the 5-kb Eco RI
fragment
which contains the p(CCG) repeat. In the first-stage
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