Appeal No. 1996-2442
Application 08/062,021
APPENDIX
1. An aqueous composition buffered to a pH of from about 7 to about 9,
which comprises:
(a) first and second primers which are specific to and hybridizable with ,
respectively, first and second nucleic acid sequences which are in opposing strands of
human cytomegaloviral DNA (hCMV DNA) and which are separated from each other
along said opposing strands from 90 to 400 nucleotides,
(b) third and fourth primers which are specific to and hybridizable with,
respectively, third and fourth nucleic acid sequences which are in opposing strands of a
second target DNA which is the same as or different from hCMV DNA, the third and
fourth nucleic acid sequences being different from said first and second nucleic acid
sequences and being separated from each other along the opposing strands by from 90
to 400 nucleotides,
each of said first, second, third and fourth primers having a Tm within the range
of from about 65 to about 74°C, all of said primer Tm’s being within about 5°C of each
other, said first and second primers having nucleotide lengths which differ from each
other by no more than 5 nucleotides, and said third and fourth primers having
nucleotide lengths which differ from each other by no more than 5 nucleotides, and
each of said first, second, third and fourth primers being present in the same amount
within the range of from about 0.1 to about 2 :molar, and
(c) a thermostable DNA polymerase present at at least 10 units/100 :l.
22. A method for the amplification and detection of human cytomegaloviral
DNA and a second target DNA comprising:
A) simultaneously subjecting the denatured opposing strands of hCMV DNA
and the denatured opposing strands of a second target DNA to polymerase chain
reaction in the presence of:
i) an aqueous composition buffered to a pH of from about 7 to about
9, and comprising
first and second primers, which are specific to and hybridizable
with, respectively, first and second nucleic acid sequences which are in opposing
1
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