Appeal No. 95-3606
Application 07/827,691
c) “a PCR reaction mixture which is substantially free of
added GTP or dGTP.”
In Fig. 1. (A), Kremer describes the following (Kremer,
p. 1712):
DNA sequence of 1.03-kb Pst I fragment containing the
p(CCG) trinucleotide repeat. . . . For sequencing, the
n
1.03-kp Pst I restriction endonuclease 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 direction led us to use
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double-stranded sequencing of pfxa2 using oligo-
deoxyribonucleotide 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.
Based on Fig. 1 and Kremer’s teaching at column 1, paragraph
bridging columns 1 and 2, to column 4, paragraph bridging
columns 3 and 4, we find that Kremer describes a method for
sequencing a 1.03-kb Pst I PCR DNA fragment containing the
p(CCG) fragile X trinucleotide repeat using at least one PCR
n
primer capable of hybridizing to nucleic acid sequences
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