Appeal No. 1996-2442
Application 08/062,021
strands of hCMV DNA and which are separated from each other along said opposing
strands by from 90 to 400 nucleotides,
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
ii) the following additional PCR reagents: a thermostable DNA
polymerase present in an amount of at least 10 units/100 :l, a DNA polymerase
cofactor and at least one dNTP, any or all of said additional PCR reagents being in the
same or a different composition as defined in i),
to simultaneously amplify said opposing hCMV DNA strands and
the opposing second target DNA strands wherein, in each PCR cycle, priming and
primer extension are carried out at the same temperature within the range of from about
62 to about 75°C and carried out within 120 seconds,
B) capturing one of said amplified hCMV DNA strands with a capture reagent
comprising a water-insoluble support to which is covalently attached a capture probe
which is specific to a nucleic acid sequence of said hCMV DNA strand, said capture
probe having from 10 to 40 nucleotides and a Tm greater than about 50°C, and is
hybridizable with said nucleic acid sequences of said hCMV DNA strand at a
temperature in the range of from about 40 to about 55°C, and
capturing one of said amplified second target DNA strands with a second
capture reagent comprising a second capture probe specific to a nucleic acid sequence
of said second target DNA strand, said second capture probe having from 10 to 40
nucleotides and a Tm greater than about 50°C, and is hybridizable with said nucleic acid
sequence of said second target DNA strand at a temperature in the range of from about
40 to about 55°C,
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