RAPID AMPLIFICATION AND DETECTION OF NUCLEIC ACIDS

AMPLIFICATION ET DETECTION RAPIDES DES ACIDES NUCLEIQUES

English Abstract

ABSTRACT
Disclosed herein are methods, primers, probes, and
kits for the rapid amplification and detection of nucleic
acids. In a preferred embodiment, a target nucleic acid
sequence in a sample is amplified by: 1) adding nucleoside
triphosphates, primer pairs comprising two oligonucleotide
primers, and a nucleic acid polymerase to the sample; 2)
denaturing the target nucleic acid sequence to form
separate strands; and 3) maintaining a reaction temperature
in a range from 68°C to 80°C and appropriate reaction
conditions wherein the following cycle occurs: the primers
hybridize to the target strands, primer extension products
are formed, the extension products separate to become
templates for the primers, and new extension products are
formed. In an alternative embodiment, the amplification
occurs at two different temperature ranges. Primer
extension products are formed in a temperature range from
68°C to 82°C, and the products are separated by raising the
temperature to range of 88°C to 96°C. Detection of the
amplified sequences occurs by using some biotin-labeled
nucleoside triphosphates, which produces nucleotide
sequences that are copies of the target sequence and that
contain one or more biotin-labeled nucleotides. The
amplified sequences are detected by contacting the sample
with immobilized probes and then detecting the presence of
the biotin. A two-stage amplification process is also
provided wherein: 1) the probe-target sequence complexes
are contacted with a first moiety that binds to biotin; 2)
a second moiety, comprising biotin bound to a detectable
moiety is added, wherein the biotin in the second moiety
binds to the first moiety; and 3) detecting or measuring
the detectable moiety. In a particularly preferred

embodiment, primer pairs and a probe are disclosed for use
when the target nucleic acid is HIV-1 DNA.

Claims

- 35 -
WE CLAIM:
1. A method for amplifying a target nucleic acid
sequence in a sample, wherein said target sequence consists
of two complementary strands, comprising the steps of:
(a) adding nucleoside triphosphates, primer
pairs comprising two oligonucleotide primers, and a nucleic
acid polymerase to said sample;
(b) denaturing said target nucleic acid
sequence to form separate strands; and
(c) maintaining a reaction temperature in a
range from about 68°C to about 80°C and appropriate
reaction conditions wherein the following cycle occurs:
said primers hybridize to said strands of said target
sequence, primer extension products, which are hybridized
to said strands, are formed from said primers and said
nucleoside triphosphates, said extension products separate
from said strands to become templates for said primers, and
new primer extension products are formed.
2. The method of claim 1 wherein said target
sequence is denatured by heating said sample to about 95°C.
3. The method of claim 1 wherein the reaction
temperature in step (c) is maintained at about 75°C.
4. The method of claim 1 wherein said primers
bind strongly to the strands of said target sequence and
the extensions from said primers bind weakly to the strands
of said target sequence.

- 36 -
5. The method of claim 4 wherein the ratio of G
and C to A and T in said primers ranges from about 1.5:1 to
about 3.0:1.
6. The method of claim 5 wherein said ratio is
about 3.0:1.
7. The method of claim 5 wherein the length of
said primers is about 15-25 nucleotides.
8. The method of claim 5 wherein the
concentration of one of said primers in said primer pair
exceeds the concentration of the other primer by a factor
of about 103 to 1 to about 102 to 1.
9. The method of claim 1 wherein the cycle of
step (c) is repeated about 10 to about 25 times.
10. The method of claim 1 wherein the reaction
temperature and conditions of step (c) are maintained for a
time from about 1.0 hours to about 2.5 hours.
11. The method of claim 1 wherein said nucleic
acid is AIDS-related viral nucleic acid.
12. The method of claim 1 wherein said target
nucleic acid is RNA and said sample is treated with reverse
transcriptase.
13. The method of claim 1 wherein said target
nucleic acid is DNA and said nucleoside triphosphates are
deoxynucleoside triphosphates.

- 37 -
14. The method of claim 13 wherein said DNA is
HIV-1 DNA.
15. The method of claim 14 wherein said target
sequence is the nucleotide sequence 1317-1379 of pHXB2.
16. The method of claim 15 wherein said primer
pair comprises the nucleotide sequences
5' GAAGGAGCCA CCCCACAAG 3' (SEQ ID N0: 1)
3' CCCCCCTGTA GTTCGTCGG 5' (SEQ ID NO: 2)
17. A method for detecting or measuring a target
nucleic acid sequence in a sample, wherein said target
sequence consists of two complementary strands, comprising
the steps of:
(a) amplifying said target sequence according
to claim 1, wherein some of said nucleoside triphosphates
are biotin-labeled, thereby producing nucleotide sequences
that are copies of said target nucleotide sequence and
contain one or more biotin-labeled nucleotides;
(b) rendering said nucleotide sequences
single-stranded;
(c) contacting said sample containing said
single-stranded nucleotide sequences with immobilized
probes, each of which comprises a single-stranded
polynucleotide attached to a solid support and capable of
hybridizing with one of said single-stranded nucleotide

- 38 -
sequences, far a sufficient time and under appropriate
hybridizing conditions to permit said polynucleotides to
hybridize with said single-stranded nucleotide sequences,
thereby forming bound complexes of said polynucleotides and
said single-stranded nucleotide sequences; and
(d) detecting or measuring the presence of
biotin in said bound complexes.
18. The method of claim 17 wherein said solid
support is a microtiter plate.
19. The method of claim 17 wherein said detecting
or measuring step comprises the steps of:
contacting said bound complexes with a
detectable moiety that binds to biotin; and
detecting or measuring said detectable moiety.
20. The method of claim 19 wherein said detectable
moiety is avidin-horseradish peroxidase.
21. The method of claim 17 wherein said detecting
or measuring step comprises the steps of:
contacting said bound complexes with a first
moiety that binds to biotin;
adding a second moiety comprising biotin bound
to a detectable moiety, whereby the biotin in said second
moiety binds to said first moiety; and

- 39 -
detecting or measuring said detectable moiety.
22. The method of claim 21 wherein said first
moiety is avidin and said detectable moiety is horseradish
peroxidase.
23. The method of claim 19 wherein said nucleic
acid is HIV-1 DNA and said single-stranded polynucleotide
of said probe comprises the sequence
<IMG> (SEQ ID NO: 3) or
<IMG> (SEQ ID NO: 6).
24. A method for amplifying a target nucleic acid
sequence in a sample, wherein said target sequence consists
of two complementary strands, comprising the steps of:
(a) adding nucleoside triphosphates, primer
pairs comprising two oligonucleotide primers, and a nucleic
acid polymerase to said sample;
(b) denaturing said target nucleic acid
sequence to form separate strands;
(c) maintaining the reaction temperature in a
range from about 68°C to about 82°C and appropriate
reaction conditions to form primer extension products from
said primers and said nucleoside triphosphates, wherein
said extension products are hybridized to said strands;

- 40 -
(d) separating said extension products from
said strands by raising the temperature to a range of about
88°C to about 96°C to produce single-stranded molecules;
(e) lowering the reaction temperature to a
range from about 68°C to about 82°C and maintaining
appropriate reaction conditions to synthesize primer
extension products from said primers and said nucleoside
triphosphates using the single-stranded molecules produced
by step (d) as a template; and
(f) repeating steps (d) and (e) a sufficient
number of times to obtain the desired amplification of said
target nucleic acid sequence.
25. The method of claim 24 wherein the temperature
at which step (d) is performed is about 90°C and the
temperature at which steps (c) and (e) is performed is
about 70°C.
26. The method of claim 24 wherein said primers
bind strongly to the strands of said target sequence and
the extensions from said primers bind weakly to the strands
of said target sequence.
27. The method of claim 26 wherein the ratio of G
and C to A and T in said primers ranges from about 1.5:1 to
about 3.0:1.
28. The method of claim 24 wherein the reaction
time for steps (c)-(f) is from about 0.5 hours to about 3
hours.

- 41 -
29. The method of claim 24 wherein said nucleic
acid is HIV-1 DNA and said nucleoside triphosphates are
deoxynucleoside triphosphates.
30. The method of claim 29 wherein said target
sequence is the nucleotide sequence 1317-1379 of pHXB2.
31. The method of claim 30 wherein said primer
pair comprises the nucleotide sequences
<IMG> (SEQ ID NO: 1)
<IMG> (SEQ ID NO: 2)
32. The method of claim 29 wherein said target
sequence is the nucleotide sequence 331-530 of pHXB2.
33. The method of claim 32 wherein said primer
pair comprises the nucleotide sequences
<IMG> (SEQ ID NO: 4)
<IMG> (SEQ ID NO: 5)
34. A method for detecting or measuring a target
nucleic acid sequence in a sample, wherein said target
sequence consists of two complementary strands, comprising
the steps of:
(a) amplifying said target sequence according
to claim 25, wherein some of said nucleoside triphosphates
are biotin-labeled, thereby producing nucleotide sequences

- 42 -
that are copies of said target nucleotide sequence and
contain one or more biotin-labeled nucleotides;
(b) rendering said nucleotide sequences
single-stranded;
(c) contacting said sample containing said
single-stranded nucleotide sequences with immobilized
probes, each of which comprises a single-stranded
polynucleotide attached to a solid support and capable of
hybridizing with one of said single-stranded nucleotide
sequences, for a sufficient time and under appropriate
hybridizing conditions to permit said polynucleotides to
hybridize with said single-stranded nucleotide sequences,
thereby forming bound complexes of said polynucleotides and
said single-stranded nucleotide sequences; and
(d) detecting or measuring the presence of
biotin in said bound complexes.
35. The method of claim 34 wherein said detecting
or measuring step comprises the steps of:
contacting said bound complexes with a
detectable moiety that binds to biotin: and
detecting or measuring said detectable moiety.
36. The method of claim 34 wherein said nucleic
acid is HIV-1 DNA and said single-stranded polynucleotide
of said probe comprises the sequence
<IMG> (SEQ ID NO: 3)

- 43 -
37. A method for detecting or measuring the
presence of a target nucleic acid sequence in a sample
comprising the steps of:
(a) amplifying said target nucleic acid
sequence through the formation of primer extension products
that contain one or more biotin-labeled nucleotides;
(b) rendering said primer extension products
single-stranded:
(c) contacting said sample containing said
single-stranded sequences with immobilized probes, each of
which comprises a single-stranded polynucleotide attached
to a solid support and capable of hybridizing with one of
said single-stranded nucleotide sequences, for a sufficient
time and under appropriate hybridizing conditions to permit
said polynucleotides to hybridize with said single-stranded
nucleotide sequences, thereby forming bound complexes of
said polynucleotides and said single-stranded nucleotide
sequences;
(d) contacting said bound complexed with a
first moiety that binds to biotin;
(e) adding a second moiety, comprising biotin
bound to a detectable moiety, wherein the biotin in said
second moiety binds to said first moiety: and
(f) detecting or measuring said detectable
moiety.

- 45 -
<IMG> (SEQ ID NO: 6).
44. A kit for detecting or measuring the presence
of a target nucleic acid sequence in a sample suspected of
containing said sequence comprising, in a container:
(a) biotin-labeled nucleoside triphosphates
and
(b) a primer pair consisting of two
oligonucleotide primers, each primer comprising an
oligonucleotide having a region that is complementary to
and hybridizes with a different strand of said target
sequence and being effective as a primer for nucleoside
polymerization.
45. The kit of claim 44 further comprising a probe
that is complementary to and hybridizes with said target
sequence.
46. The kit of claim 45 further comprising means
for detecting or measuring said biotin.
47. The kit of claim 46 further comprising a
nucleic acid polymerase.
48. The kit of claim 44 wherein said primer pair
comprises the nucleotide sequences
<IMG> (SEQ ID NO: 1)
<IMG> (SEQ ID NO: 2)

- 46 -
49. The kit of claim 44 wherein said primer pair
comprises the nucleotide sequences
<IMG> (SEQ ID NO: 4)
<IMG> (SEQ ID NO: 5)
50. The kit of claim 45 wherein said probe
comprises the nucleotide sequence
<IMG> (SEQ ID NO: 3)
51. A kit for detecting or measuring the presence
of a target nucleic acid sequence in HIV-1 DNA in a sample
suspected of containing said sequence comprising, in a
container:
(a) biotin-labeled nucleoside triphosphates:
(b) a primer pair comprising the nucleotide
sequences
<IMG> (SEQ ID NO: 1)
<IMG> (SEQ ID NO: 2)
and
(c) a probe comprising the nucleotide
sequence
<IMG> (SEQ ID NO: 3)

Description

7 ~3
RAPID AMPLIFICATION AND DET~CTION OF NUCLEIC ACIDS
FIELD OF THE INVENTION
This invention relates to the rapid amplification
and detection of nucleic acids. In particular, the
invention provides improved methods for amplifying small
amounts of nucleic acids in a sample in which the
amplifcation ~teps are conducted at the same tempexature
or, alternatively, at only two different temperatures. In
addition, the invention provides improved methods ~or
detecting the ampliied nucleic acids in which the
detection signal is boosted. Related probes and test kits
are also provided. The invention is expected to be useful
in a wide variety of fields, including scientific,
clinical, and forensic analysis. In one specific
embodiment, it is particularly useful in the detection of
human immunodeficiency Yirus type 1 (HIV-l), the causative
agent of AIDS.
BACKGROUND OF THE INVENTION
The ability to detect exceeding small amounts of a
nu-leic acid in a sample generally requires the
amplification of the amount of the target nucleic acid.
This is especially important for the detection of human
retroviruses, where positive samples may contain only 5-10
target molecules in 106 cells.
The preferred method for amplifying target DNA has
been the polymerase chain reaction (PCR) techni~le. The
technique has been described in U.S. Patent Nos. 4,683,195,
issued July 28, 1987 to Mullis, et al., 4,683,202, issued
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July 28, 1~87 to Mullis, 4,800,159, issued January 24, 1989
to Mullis, et al., 4,889~818, issued December 26, 1989 to
Gelfand, et al., and 4,902,624, issued February 20, 1990 to
Columbus, et al., all of which are incorporated herein by
reference.
In general, the PCR reaction involves the use of a
pair of specific oligonucleotide primers to initiate DNA
synthesis on a target DNA template. Two oligonucleotide
primers are used for each double~stranded seyuence to be
amplified. The target sequence is denatured into its
complementary strands. Each of the primers, which are
sufficiently complementary to a portion of each strand of
the target sequence to hybridize with it, anneals to one of
the strands. The primers are extended, using nucleosides
in the sample and a polymerization agent, such as heat-
stabl ~g DNA polymerase. This results in the formation
of complementary primer extension products, which are
hybridized to the complementary strands of the target
sequence. The primer extension products are then separated
from the template strands, and the process is repeated
until the desired level of ampli~ication is obtained. In
subsequent cycles, the primer extension pxoducts serve as
ne~ templates for synthesizing the desired nucleic acid
sequence.
By repeating the cycles of denaturation, annealing,
and extension, the original target DNA can be ampli~ied
exponentially according to the formula 2n, where n is the
number of cycles. In theory, 25 cycles, for example, would
result in a 3.4 x 107-fold amplification. However, since
the efficiency of each cycle is less than 100%, the actual
amplification after 25 cycles is about 1-3 x 105-fold. The
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- 3 - 2 ~
size of the amplified region i5 generally about 100-400
base pairs, although stretches of up to 2 kb can be
amplified. See Xeller and Manak, DNA Probes (Mew York:
Stockton Press, 1989), pgs. 215-216.
The three basic steps of the PCR reaction --
denaturation, annealing, and extension -- are driven and
controlled by the temperature of the reaction mixture, with
each ~tep occurring at a different temperature. Somewhat
different temperature ranges are disclosed for each of the
three steps in the above-referenced patents. However, as
time passed, those skilled in the art have settled on
fairly standard temperatures ~or each of the steps in the~
cycle. Thus, Gelfand, et al., discloses a denaturing
temperature range of about 90--105-C, preferably 90--lOO-C,
an annealing temperature range of about 35 -65-C,
preferably 37--60 C, and an exten ion temperature range of
about 40-80C, preferably 50-75C. For ~ polymerase,
which is the overwhel~ingly preferred polymerase ~or the
PCR reation, Gelfand, et al., refers to an annealing
temperature range o~ ab~ut 45--58-C and a~ extension
temperature range of about 65--75-C. Columbus, et al.,
which is directed to a temperature cycling cuvette for use
in PCR, ref~rs to temperature ranges of 92'-95 C for the
denaturing step, 50 -60~C for the annealing step, 70C for
the extension step. Keller and Manak, cited above, refer
to a denaturation temperature of about 93 C, an annealing
temperature of 37-55 C, and a primer extension temperature
of 70-C.
The PCR technique has been modified to permit the
amplification of viral RNA. See Murakawa, et al., DNA,
7:287-295 ~1988), which is incorporated herein by
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- 4 - 2~3~g3
reference. The article discloses the amplification of
sequences from HIV-l ~NA templates for the identification
of HIV-1 in peripheral blood and tissue samples obtained
from AIDS and ARC patients. Total nucleic acid is isolated
from infected cells, and the DNA is digested with RNase-
free DNase so that it does not contribute to the final PCR
product. A cDNA copy of a target sequence of the virAl RNA
is synthesized, using the PCR primers and reverse
transcriptase. The one primer compl~mentary to the RNA
serves to initiate cDNA synthesis.
Several different formats have been used for the
detection of PCR products. Generally, a radioactive or
nonradioactive labeled probe that is complementary to the
target sequence is used. The hybridization of the probes
to the amplified target sequence, and the subsequent
detection of the labeled moiety results in the detsction of
the target sequence. Nonradioactive labeled probes are
generally more desirable because th~y obviate the n~ed for
special handling procedures. However~ they may not
generate as intense a signal, or the signal may be
obscurred by background "noise. Il Thus, there is a need for
enhancing the intensity of the signal in such probes.
The PCR technique is a revolutionary one, and it is
widely used~ Nevertheless, it does have significant
drawbacks. The most serious of these is nonspecific
hybridization, which results in false positives. Avoiding
nonspecific hybridiæation requires ultrapure reagents.
Unfortunately, for most clinical and diagnostic
applications, it is desirable to use "dirty" samples, which
presents a major problem, unless time-consuming and
expensive sample preparation is undertaken.
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Another important drawback to the PCR technique is
the time involved in amplification. Although the usual six
hour time period for PCR is far superior to an alternative
technique such as cloning, which can take days or weeks, it
would still be desirable to cut amplification time by one-
half to two-thirds.
The present invention overcomes these draw~acks of
the PCR technique, and it provides an improved detection
system. The invention provides methods for the rapid
amplification and detection of nucleic acids in which the ~
denaturing, annealing, and extension steps occur all at the
same telmperature or, alternatively, at only two dif~erent
temperat:ures, thus providing for faster cycling. In
addition, the invention provides amplification methods
where th,e annealing temperature is higher than the prior
art temperatures, thus eliminating nonspecific
hybridization. Finally, the invention provides improved
methods of detecting the amplified nucleic acids through a
two-stage signal amplification.
SUMMARY OF THE INVENTION
It is an object o~ the present invention ~co provide
me~hods for amplifyinq a target nucleic acid sequence in a
sample.
Another object is to provide methods for detecting
or measuring the presence of a target nucleic acid sequence
in a sample.
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- 6 ~ 3
Still another object of the invention is to provide
a kit for detecting or measuring the presence of a target
nucleic acid sequence in a sample.
A further obj~ct of the inventlon is to provide
primer pairs for use in amplifying a nucleic acid sequenc~
~ of HIV-l DNA.
; A still further object is to provide a probe for
use in detecting a nucleotide sequence complementary to a
target nucleic acid sequence of HIV-l DNA.
.
Additional objects and advantages of the invention
will be set forth in part in the description that follows,
and in part will be obvious from the description, or may be
learned ~y the practice of the invention. The objects and
advantages of the invention will be attained by means of
the instrumentalities and combinations particulary pointed
out in the appended claims.
To achieve the objects and in accordance with the
purpose of the invention, as embodied and broadly described
herein, the present invention provLdes a method for
amplifying a target nucleic acid sequence in a sample. The
sequence is part of a nucleic acid, which the sample is
suspected of containing. Pre~erably, the target nucleic
acid is DNA, most preferably HIV-l DNA.
Nucleoside triphosphates, primer pairs consisting
of two oligonucleotide primers, and a nucleic acid
polymerase are added to the sample. Each primer is an
oligonucleotide having a region that is complementary to
and hybridizes with a different strand of the target
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- 7 ~ 2~7~
sequence. It is effective as an initiator for nucleoside
polymerization. If doubls stranded, the nucleic acid in
the sample is denatured so that separate strands of the
target nucleic acid sequence are formed. Preferably, the
denaturing is accomplished by heating the sample to about
95C.
A reaction temperature in a range from about 68C
to about 80C (preferably about 75C) and appropriate
reaction conditions are maintained so that the following
cycle occurs. First, the primers anneal (hybridizeJ to the
separate strands of the target sequence. The primers and
the nucleic acid polymerase initate the synthesis of primer
extension products, formed by the nucleosides attaching to
the primer and forming a polymer, using the target sequence
strand as a template. Thus, the primer extension products
are annealed or hybridized to the strands. The primer
extension products are then separated from the strands to
become templates for the primers, and the cycle is
repeated, with new primer extensio~ products being formed.
The reaction is allowed to continue for a sufficient number
of cycles until the desired amplification of the target
nucleic acid sequence has been accomplished. The primers
are chosen so th~t they bind strongly to the strands of the
target seguence, but the extension from each primer binds
weakly to the strand.
In an alternative embodiment of the invention, the
cycling occurs at two different temperatures. The reaction
temperature is maintained in a range from about 68-C to
about 82-C (preferably about 70-C) and appropriate reaction
conditions are maintained to form the primer extension
products. These are separated from the strands by raising
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- 8 - 2~ 3
the temperature to a range of about 88'C to about 96C
(preferably about 90C) to produce single-stranded
molecules. The temperature is then lowered back to the
previously mentioned range, permitting the primers to
anneal to the single-stranded molecules and primer
extension products to be synthesized, using the single-
stranded molecules as templates. The temperature cycling
is repeated a suf~icient number of times to obtain the
desired amplification of the target nucleic acid sequence.
The invention also provides methods for detecting
or measuring a target nucleic acid sequence in a sample,
based upon the amplification methods discussed above. In
these methods, some of the nucleoside triphosphates are
biotin-labeled. This produces nucleotide sequences that
are copies of the target sequence which contain one or more
biotin-labeled nucleotides. Such sequences are rendered
sin~le-stranded and contacted with immobilized probes.
Each of the probes is a single-stranded polynucleotide
attached to a solid support and capable of hybridizing with
one of the single-stranded nucleotide sequences. The
probes are contacted with the sample for a su~ficient time
and under appropriate hybridizing conditions to permit the
polynucleotides to hybridize with the single stranded
nucleotide sequences. This forms bound complexes comprised
of both of these entities. The presence of the biotin in
the bound complexes is then detected or measured.
Preferably, the biotin is detected by contacting the bound
complexes with a detectable moiety that binds to biotin,
such as avidin-horseradish peroxidase, and then detecting
the detectable moiety, i.e., the horseradish peroxidase.

9 2~7~3
;~ In an alternative and pref~red embodiment, the
detecting step is accomplished by contacting the bound
complexes with a first moiety that binds to biotin, such as
avidin. A second moiety, comprising biotin bound to
detectable moity, such as horseradish peroxidase, is then
added. The biotin in this second moiety binds to the first
moiety. The detectable moiety is then detected. This
provides a two-stage amplification of the signal.
The invention ~urther provides kits for detecting
or measuring the presence of a target nucleic acid sequence
in a sample. The kits contain biotin-labeled nucl~oside
triphosphates and a primer pair speci~ic for the particular
target sequence sought to be detected or measured.
Preferably, the kit further comprises a probe that is
specific for the target sequence. It may also contain a
nucleic acid polymerase, reagents for detecting or
measuring the biotin, denaturing reagents, and/or controls.
In a preferred emhodiment of the Xit, the targ~t
nucleic acid sequence is HIV-1 DNA. The primer pair
comprises th~ nucleotide sequences
5' GAAGGAGCCA CCCCACAAG 3' (SEQ ID NO: 1)
3' CCCCCCTGTA GTTCGTCGG 5' (SEQ ID NO: 2)
and the probe comprises the nucleotide sequence
5' TTTAAACACC ATGCTAAACA CAGT 3'. (SEQ ID N9: 3)

2 ~ 3
-- 10 --
DETAILED DESCRIPTION OF THE INVENTION
Reference will now made in detailed to the
presently preferred embodiments of the invention which,
together with the following examples, serve to explain the
principles of the invention.
The invention provides improved methods for
amplifying and detecting nucleic acids. The target nucleic
acid may be single or double-stranded DNA or RNA from any
organism. Such organisms include plants, animals, and
microorganisms, such viruses, viroids, mycoplasma,
bacteria, and fungi. The viruses include DNA and RNA
viruses. In a pre~erred embodiment, the viruses are
retroviruses, and in a particularly preferred embodiment,
they are the AIDS-related viruses (including HIV-l and HIV-
2). The term "animals" includes ma~mals, and, in a
preferred embodiment, the target nucleic acid is mammalian
nucleic acid, such as mitochondrial or genomic DNA or the
various types of RN~. Such nucleic acid further includes
human cellular oncogene sequences and human structural gene
sequences.
The sample may he anything that contains nucleic
acid, obtained from a source by techniques known to those
skilled in the art. It may be further processed by known
techniques, such as being subjected to extractio~
procedures, to render it in a form usable in the method of
the invention. For examplel the method of the present
invention can be applied to determine if a patient has been
infected by a virus. A sample of body fluid or tissue that
contains the virus is obtained from the patient. In this
case, the sample should contain cells that are capable of
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207aO83
being infected by the virus or should contain body fluid in
which the virus or viral nucleic acid is known to
accumulate. The viral nucleic acid is extracted from the
sample by known techniques~
A target nucleotide sequence within the target
nucleic acid i5 selected so as to provide a target of
sufficient specificity and uniqueness in order to be
identified and distinguished from other nucleic acid
molecules in the sample. Among other things, it will be
selected on the basis of a nucleotide base sequence
specific to the organism. For many viruses, in particular
those that mutate ~t high rates, it is advisable to select
a target sequence from a well-cons~rved region of the viral
genome.
The targst nucleotide se~lence may consist of from
about 50 to about 4000 base pairs, but preferably consists
of about 55 to about 200 base pairs, and most preferably
consists of about 60 to about lOO base pairs. In the case
; of HIV~l DNA, the preferred target: sequences are the
nucleotide sequence 1317-1379 of pHXB2 and 331-530 of
p~XB2, depending upon which primer pair of the invention is
used to amplify the sequence.
; In the preferred embodiment of the invention, the
target sequence is amplified in a reaction that occurs at a
single temperature, after the initial denaturation of the
nucleic acid, in contrast to the three different
temperatures in the PCR technique. W~ call this method the
Continuous Enzyme Reaction (CER). In the first step,
certain reagents are added to the sample that contains the
target nucleic acid. In particular, nucleoside
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- 12 ~ r7 ~ ~ 3
triphosphates, including biotinylated nucleotide
triphosphates, primer pairs, and a nucleic acid polymerase
are added. Preferably, they are added as part of a
buffered solution. Each primer is an oligonucleotide
having a region that is complementary to and hybridizes
with a different strand of the target sequence. It is
effective as an initiator for nucleoside polymerization.
Preferably, the length of each primer is approximately 15-
25 nucleotides.
The target nucleic sequence, if it is double-
stranded, is then separated into single strands by known
techniques. Preferably, the nucleic acid is denatured by
heating the sample to about 95C. The target sequence may
be denatured before the other reagents are added to the
sample, although it is preferable to denature it
afterwards.
The sample is then maintained under appropriate
reaction conditions to permit the following cycle to occur:
(1~ each of the oligonucleotide primers anneals
(hybridizes) to each of the strands of the denatured target
sequence; (2) primer extension products are synthesixed
from the nucleoside triphosphates, with the systhesis being
initiated by the primer and catalyzed by the nucleic acid
polymera~e; and (3) a portion of the extension products,
which are hybridized to the strands of the target sequence,
separate spontaneously from the strands. The separated
extension products become templates for the primers, and
the cycle is repeated. Such reaction conditions include
maintaining the reaction temperature som~where in the range
from about 68C to about 80 C. Preferably, the temperature
is maintained at about 75C.
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- 13 ~ 2 ~ 7 e~ O g c~
The reaction is permitted to proceed for a
suf f icient period of time so that the cyclP is repeated
about 10 to about 25 times. This will occur over a time
ranging from about 1.0 hours to about 2.5 hours.
Generally, the longer reaction time is preferred.
The nucleoside triphosphates are readily available
commercially or c~n ~e synthesized by those skilled in the
art. When the method of the inv~ntion is applied to the
amplification of DNA, deoxyribonucleoside triphosphates axe
usedO
Most preferably, some of the nucleoside
triphosphates or deoxyribonucleoside triphosphates are
labeled with biotin. This permits the incorporation of a
reporter molecule into the amplified sequences, which can
be detected by the means described below. Such biotin~
labeled nucleoside triphosphates can be synthesized by
known ~echniques or are commercially available. The
preferred biotin-labeled nucleoside triphosphate is Bio-
dUTP. The labeled nucleosides are used in a concentration
of about 10 uM to about 5mM, and preferably from about
O.1 mM to about 0.5 mM.
The polymerizing agent is any polvmerase for
nucleic acid. Preferably, it is Taq polymerase, which is
heat stable under these reaction conditions.
The primer pairs consist of two oligonucleotide
primers. Each primer is an oligonucleotide having a region
that is complementary to and hybridizes with a different
strand of the target sequence, and is effective as an
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- 14 - 2~7~3
initiator (primer) for nucleoside polymerization. The
primers of the invention are selected so as to meet two
requirements. First, they must bind strongly to the
strands of the target sequence to allow binding at a
temperature higher than the temperature preferred in the
literature. Second, the product from the extension of the
primers, which is synthesized from the nucleosides in the
presence of the polymerase, is the least stable of a series
of products which were initially selected and more likely
to dissociate spontaneously to single-stranded form, thus
itself becoming a target sequence of the appropriate
primer. As used herein, the term "bind strongly" and
variations thereof means adequately stable single-stranded
16DNA-primer hybrids are formed at the high temperature of
the in~ention.
This is accomplished by choosing primers where the
ratio of G and C to A and T in the primers ranges from
about 1.5:1 to about 3.0:1. Preferably, the ratio is about
3.0:1. This is confirmed by the temperature of
dissociation (Td) of the primer pair and the meltin~
temperature (Tm) of the extension products. The Td of the
primer pair is calculated from the formula Td = 4 D (C+G) +
2(A~T). The Tm, which is the temperature at which the
extension products are denatured, is calculated from the
formula:
Tm = 81.5D + 16.6 log Ci + 0.41 (% C+G) - 0.72 (%
formamide) - 82/n - 1.5 (% mismatch)
where Ci is the ionic strength of the reaction solution and
n is the number of nucleotides.
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For HIV-l DNA, the following specific primers,
which we call HOPE l and HOPE 2, are preferred:
HQPE 1: 5' GAAGGAGCCA CCCCACAAG 3' (SEQ ID NO: l~
HOPE 2: 3' CCCCCCTGTA GTTCGTCGG 5' (SEQ ID NO: 2)
These primers produce HIY-l DNA sequences in positions
1317-1379 of the plasmid pHXB2, which are 63 base pairs
long. The Td for HOPE 1 is 62C and that for HOPE 2 i5
64C. The Tm of the product, i.e., the primer extension,
is 76.7C.
The use of primers meeting the above-stated
conditions permits the maintenance of temperature and other
reaction conditions whereby the amplification cycle occurs
at a single temperature rather than three different
temperatures. In order for the reaction to occur
efficiently in terms of yielding double stranded target
DNA, the following reaction con~itions should be
considered:
1~ The temperature sho~ld be high enough so that
a substantial portion of th~ target DNA is found in single-
stranded form, available for primer hybridization ~the
activity of the polymerase is a limiting factor).
2) The concentration of the primers should be
high enough so as to favor single-stranded DNA-primer
hybrid formation.
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2~7~3
~ 16 -
3) The GC content of the primers should be such
as to allow an adequately stable single-stranded DNA-primer
hybrid to be formed at the high temperature chosen.
4) The primer extension product, on the contrary,
should be as unstable as possible so that the probability
for the two strands to dissociate spontaneously is as high
as possible.
5) The polymerizing agent should be able to
efficiently polymerize dNTPs at the high temperature chosen
and also to incorporate the dNTP analogue used as a label
onto DNA strands.
Generally, the cycle of primer annealing, primer
extension, and extension product denaturing occurs from
about 10 to about 25 times~ The time period for this to
occur is approximately 0.5 hours to overnight.
In an alternative and preferred embodiment,
asymmetric concentrations of the primers are used. That
is, the concentration of one of the primers exceeds that of
the other by a factor of about 103 to 1, and preferably by
about 102 to about 1. This permits the build up of only
one instead of two primer extension products. Therefore,
there is only a limited risk of DNA-DNA complexing during
hybridization. Instead, the product DNA will bind to the
primer or the probe that is used for detection of the
amplified DNA. This enhances both the specificity and
sensitivity of the assay.
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When the nucleic acid to be amplified is RNA,
certain modifications are made. The RNA template in
infected cells is either viral mRNA or packaged virion RNA.
Total nucleic acid is isolated in crude form by known
methods, or it can be purified by, for example, phenol-
chloroform extraction. Residual DNA may be digested with
RNase-free DNase sv that it does not contribute to the
amplified sequences. A cDNA copy of a target
ribonucleotide sequence in the target RNA is synthesized,
using the primers and reverse transcriptase. The primer
complementary to the target sequence serves to initate cDNA
synthesis. The single stranded cDMA product can be
amplified after heat inactivation of the reverse
trans~riptase and adjustment of the reaction conditions to
those of the method of the in~ention.
In an alternative embodiment of the invention, the
amplification of the target nucleic acid sequence occurs at
two different temperatures. The ~primer annealing and
extension steps occur at one temp'erature, and the
denaturing of the primer extension products occurs at
another temperature. We call this method the Accelerated
Ch~in Enzyme Reaction tACER).
In particular, the reaction temperature is
maintained at a point in the range from about 68OC to about
82-C for primer annealing and extension. Preferably, the
reaction temperature for these steps of the cycle i8 about
70C. The primer extension products are separated from the
target sequence strands or other extension products by
raising the temperature to a range of about 88C to about
96C. This denatures the extension products from the
template, producing single-stranded mOlecules that serve as
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further templates when the temperature is lowered.
Preferably, the denaturing step occurs at a temperature of
about 90C.
The cycling between these two temperatures is
repeated about 10 to about 25 times. The reaction time for
these steps is from about 0.5 hours to about 3 hours.
Generally, the time is about 1.5 hour~.
The preparation of the sample and the initial
reaction mixture is essentially the same as in the single
temperature method. The target nucleic acid sequence is
also denatured in the same manner. This method also lends
itself to the use of asymmetric concentrations of primers
and to the amplification of RNA as discussed previously.
The specific primers for any given target sequence
are determined ~s discussed above with respect to the
single temperature e~bodiment. The primers will have the
same ~eneral chararteristics mentioned above. For HIV-1
DNA, the previously mentioned HOE'E 1 and HOPE 2 primers are
preferred. In addition, two other primers, which we
designate NIDA l and NIDA 2, are preferred. These primers
comprise th~ following sequences:
NIDA 1: 5' GACATCGAGC TTGCTAGAAG 3' (SEQ ID NO: 4)
NIDA 2: 3' GGTGACGAAT TCGGAGTTAT 5~ ~SEQ ID NO: 5)
They produce HIV-1 DNA sequences in positions 331-530
pHXB2 .
,

- 19 - 2~7~83
In another embodiment, the invention comprises a
method for detecting or measuring a target nucleic acid in
a sample. The target s~quence is amplified according to
the methods discussed above, wherein some of the nucleoside
triphosphates are biotin-labeled. This produces nucleotide
sequences that are copies of the target sequence and
contain one or more biotin-labeled nucleotides. The
amplified sequences, which will be double-stranded, are
then rendered single-stranded by known techniques,
preferably heat denaturation at a temperature of about 88-C
to about 96C. The sample is then contacted with
immobilized probes. These probes are single stranded
polynucleotides attached to a solid support. Each one is
capable of hybridizing with one of the single-stranded
nucleoti~e sequences. The probes are contacted with the
sample for a sufficient time and under appropriate
hybridizing conditions known to those skilled in the art to
permit the polynucleotides to hybridi~e with the single-
stranded nucleotide sequences. This forms bound complexes
of the polynucleotides and the single-stranded nucleotide
sequences.
The probes can be prepared by standard techniques
known to thase skilled in the art, given the particular
targe~ sequence and the teachings contained herein. When
the nucleic acid is HIV-l DNA, the preferred probe
comprises the sequence 5' TTTAAACACC ATGCTAAACA CAGT 3'
(SEQ ID NO: 3), which we call the HOPE 3 probe. The ~OPE 3
probe may also be used with a pyrimidine spacer at the
5'end. Preferably, the spacer is CTCTC, in which case we
call the probe HOPE 4.
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The solid support may be any solid material to
which the probe may be attached. Such material includes
filters, resins, beads, cubes, and microtiter plates.
Preferably, the solid supports are the wells in plastic
microtiter plates, such as polystyrene microtiter plates.
Most preferably, the probe is first chemically coupled to a
protein carrier, such as bovine serum albumin, which is
then immobilized onto polystyrene microtiter plate wells.
See Running and Urdea, BioTechniques, 8:276-277 tl990) and
Nagata, et al., EBS Letters, 183:379-382 (1985), both of
which are incorporated herein by reference.
The single-stranded polynucleotide of the probe is
attached to the solid support covalently or noncovalently
by means known to those skilled in the art. Preferably,
the probe is chemically coupled to a protein, such as
bovine serum albumin, or other carrier, such as
polyethylene glycol. A spacer between the carrier and the
probe may also be used. The polynucleotide comprising the
probe may contain from about 15 t:o about 2,000 nucleotides.
Preferably, it contains from about 15 to about 200
nucleotides and most preferably from about 15 to about 40
nucleotides.
; The final step in this method of the invention is
to detect or measure the presence of biotin in the bound
complex. One way of doing so is to contact the bound
; complexes with a detectable moiety that binds to the biotin
and then to detect such moiety. Preferably, such
detectable moiety is comprised of a molPcule or compound
that binds to the biotin, such as avidin or streptavidin,
coupled to a detectable entity (e.g., a detectable molecule
or compound), such as horseradish peroxidase. The
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- 21 - ~ ~7 )~8~
horseradish peroxidase i5 detected by known means. Other
detectable moieties/detectable entities are disclosed in
V.S. patent No. 4,711,955 issued December 8, 1987 to Ward
et al., which is incorporated herein by reference.
Preferably, the bound complexes are washed with a
liquid, such as a buffer solution, before and/or after
contact ~ith the detectable moiety to remove any unbound,
labeled sequences or labeled nucleoside triphosphates that
may be present.
In a particulary preferred embodiment, ~he
invention provides a method for a two-stage amplification
of the signal provided by the biotin. The method may be
used to detect target nucleic acid se~uences that have been
amplified through the formation of primer extension
products that contain one or more biotin-lab~led
nucleotides. Thus, i~ is partl~ularly applicable to the
amplification methods o~ the present invention, but it is
not limited to them.
Such ampliPied sequences are rendered single-
stranded, and the sample containing such single-stranded
sequences is contacted with immobilized probes as discussed
above. The bound complexes comprising the single-stranded
sequences and the single-stranded polynucleotides of the
probes are then contacted with a first moiety that binds to
biotin. The moiety i5 preferably avidin or streptavidin.
A second moiety, comprising biotin bound to a detectable
moiety, is then added to the sample. The biotin in the
second moiety binds to the ~irst moiety, which is bound to
the biotin in the amplified sequences. The detectable
moiety is then detected or measured by standard techniques.
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Preferably, such detectable moiety is horseradish
peroxidase. Preferably, the solid support is washed
between the steps discussed above in order to r~move
unbound materials that may interfere with the detection
procedure or giv~ a false signal.
The present invention also comprises a kit for
detecting or measurin~ the presence o~ a target nucleic
acid sequence in a sample suspected of containing that
sequence. The kit comprises biotin-labeled nucleoside
triphosphates and an appropriate primer pair. Each primer
comprises an oligonucleotide having a region that is
complementary to and hybridizes with a di~ferent strand of
the target sequence and is effective as a pri~er for
nucleoside polymerization. Preferably, the kit further
comprises a probe that is complementary to and ~ybridizes
with the target sequence. Most pre~erably, the ki~ further
comprises a nucleic acid polymerase, preferably ~
polymerase, and additional means, such as reagents, or
detecting or measuring the biotin, denaturing double-
stranded polynucleotides, and providing a control against
which the results may be evaluated.
In a particularly preferred embodiment, the
invention provides a kit for detecting or measuring the
presence of ~ target nucleic acid sequence in HIV-1 DNA in
a sample. The kit contains biotin-labeled nucleoside
triphosphates, unlabeled nucleoside triphosphates, a primer
pair comprising the nucleotide sequences:
5' GAAGGAGCCA CCCCACAAG 3' (SEQ ID MO: 1)
3' CCCCCCTGTA GTTCGTCGG 5' (SEQ ID NO: 2)
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and a probe comprising the nucleotide sequence
5' TTTAAACACC ATGCTAAACA CAGT 3'. (SEQ ID NO: 3)
It is to be understood tha~ the application of the
teachings of the present invention to a specific problem or
environment will be within the capabilities of one having
ordinary skill in t~e art in light of the teachings
contained herein. ~xamples of the products and processes
of the present invention appear in the following examples.
EXAMPLE 1
Isolation of DNA From Blood
For AIDS screening, the object is a rapid and safe
extraction o~ DNA to allow the processing o~ many samples.
The suggested method is the following. Four hundred
microliters (400 ul) of blood is ~ollected in a tube
containing anti-clotting agent (citrate or EDTA) and is
contrifuged (e.g. 3,000 g for 5 minutes or 10,000 g for 5-
10 seconds). The upper buffy-coat layer is removed, or the
whole pellet is suspended in 200 ul distilled water in
order to lyse the cells. The lysed cell suspension is
boiled at 100C in order to break up the cell nuolei.
Centri~ugation for 5 minutes at 10,000 g remoYes insoluble
debris. The resultant supernatant is used for subsequent
manipulations.
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- 24 - ~ ~7~83
EXAMPLE 2
One Temperature Reaction (CER~
Fifteen ul of DNA sample prepared as in Example 1
are added to 80 ul of a reaction buffer. The reaction
buffer contains 0.3nmol of each primer (HOPE 1, HOPE 2),
lOmN Tris-HCl, pH 8.4 (20C~, 50mM KCl, 2mM MgC12, 0.1%
gelatin. 0.5mM dATP, dCTP, dGTP, 150 uM dTTP, 350 uM Bio-
11-dUTP, and 0.1~ BSA (nuclease free). The reaction mix is
incubated at 95'C for ~ive minutes. This is to initially
denature the DNA. The mix is then allowed to cool down to
room temperature, three units of the enzyme Vent polymerase
(New England Biolabs) are added, and mineral oil is lay~red
at the top. The reaction then takes place at 78~C for 3-4
hours.
EXA~spr.~ 3
-~;
Two Temperature Reaction (ACER!
:: .
Ten microliters of the DNA containing supernatant
prepared as in Example 1 are used for this amplification
reaction. This volume is mixed with 90 ul of reaction
bu~fer. The reaction buffer comprises 100pmol each primer
(melting temperature higher than 62-C, i.e., NIDA1, NIDA2~,
lOmM Tris-HCl, pH 8.0 (20C), 1.8mM MgC12, 50 mM K~l, 0.0~%
gelatin, 0.1mM dATP, dCTP, dGTP, 03n~ dTTP, 70nM Bio~
dUTP, and 0.1% BSA tnuclease free).
The reaction mixture is then layered with one or
two drops oP any mineral oil and transferred to a
programmable heat block where amplification takes place.
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The temperatures used are 90~C for the initial and
subsequent denaturation steps, and 70C for annealing and
elonyation. The incubation period allowed for denaturation
is one minute and for annealing, which is coupled with
elongation, two minutes. This thermal profile is repeated
20 times~ with the annealing-elongation step of the 20th
cycle being the last step of the reaction. Reaction
products are stored at 4C for ~urther manipulation. The
amplification reaction takes place in the wells of a 96-
well ELISA plate, a very convenient way when dealing with a
large number of samples.
EXAMPL_ 4
Two Temperature Asymmetric Reaction tAACER~
This type of reaction is essentially the same as
the ~CER, except for the amount of the primers, which now
is 20pmol ~or HOPE 1 and 300pmol ~or HOPE 20 The volume of
the DNA sample is also increased to 20 ul.
EXAMPL~ 5
Detection of AmpllPied_Products
The totality (100 ul~ of the amplification reaction
volume (ACER or CER) i5 applied to a well containing
i~mobilized probe after heat denaturation (lOO C, five
minutes) and snap cooling on ice. (Denaturation iR not
necessary when asymetric two temperatures, or one
temperature reactions are performed.) Hybridization is
allowed to proceed for one hour at 37-C. Unbound material
is removed by washing four times with O.OM NaCl.
. . .
.

- 26 - 2~3~83
A 50ng/ml solution of avidin-HRP in lOmM Tris-HCl
pH 7.4 (20C3, 5% gelatin, and 2~ 3SA is added to the well.
After incubating 30 minutes at room temperature with
shaking, the well is washed with O.9M NaCl, lOmM Tris-HC1,
pH 7.4 (20C), and 001% Tween-20. The substrate mix is
then applied (0.05% TetraMethylBenzidine, 10% H202). Blue
color develops within 10 minutes. The colorigenic reaction
is stopped with O.5% H2S04, and absorbence at 450nm is
measured.
EXAMPLE 6
Preparation of the Primers
- The synthesis of the primers is standard and
automated (Applied Biosystems synthesizer, model 3BOB).
SyntAesis is based on a solid phase chemistry where the
first ba~e is already bound to a controlled pore glass
(CPG) support at the 3' end. The 5' end is protected with
a di~ethoxytrityl ~roup. Each new base is covalently
coupled to the previous base at the 5' end using a complete
synthesis cycle. The cycle conslsts of 4 steps:
1. ~etritylation. The attached (terminal) base
is detritylated with trichloroacetic acid creating a free
hydroxyl group.
2. Activation and addition. The next base is
activated with tetrazole at the 3' end and then is passed
through the CPG column allowing it to react with 5'
hydroxyl of the terminal base.
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- 27 - 2Q7.~g~
Capping. The unreacted 5' hydroxyl groups
(approximately 0-2%) are blocked with acetic anhydride
(acetylation).
4. Oxidation. The phosphorous of the new base is
oxidized from the trivalent to the pentavalent form using a
iodine-water-lutidine-tetrahydofuran mixture.
After the completion of the synthesis cycle, the
new base is accessible for the repeat of the cycle with the
next base. Thus, elongation of the DNA chain i~ achieved
with the correct nucleotide sequence.
At the end of the run, the product is cleaved from
the CPG using 25~ a~monia solution. The bases of the
resultant oligonucleotide are then deprotected by heat-
treatment at 55OC in the presence of ammoni~ for 5 houxs.
The product is evaporated to dryness to remove the
ammonia, reconstituted in water and dried again, and
finally reconstituted in water. The product is stored at
-20C until further use.
EXAM~LE Z
Preparation of the Probe
The oligonucleotide HOPE4 (5' CTCTCTTTAA ACACCATGCT
AACACAGT 3') (SEQ ID NO:6) was synthesized using a DNA
synthesizer (Applied Biosystems). It was then chemically
coupled to bovine serum albumin, which was immobilized into
the wells o~ a polystyrene microtiter plate according to
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2~7~3
- 28 -
the method of Running and Urdea, op. clt., and Nagata, et
al., op. cit.
EXAMPLE 8
Evaluation of Samples
Eight seropositive samples were examined, plus a
DNA sample prepared from an infected culture of OEM cells.
Several seronegative samples were also examined. A
positive control-plasmid derived cloned sequences of HIV-I,
and a negative control, were always included.
Some samples were analyzed by applying CE~ and
others by ACER. AmpliPication products were detected by
either in solution hybridization with a specific
radiolabeled probe tHOPE 3) and ~ubsequent acrylamide gel
electrophoresis or the ELISA-like colorimetric assay using
as label a biotinylated dNTP analog or a labeled primer
(HOPE 2).
The vaIues obtained were as follows:
Absorbance readings of ELISA-like assay:
1 POS1 0.40 seropositive (5 yrs)
2 POS) 0.30 seropositive (1 yr)
3 POS) 0.60 dead
4 POS) 0.20 seropositive (1.5 yrs)
5 POS) 0.40 under AZT (4 yrs)
6 POS) 0.55 dead
7 POS~ 0.30 seropositive (2 yrs)
8 POS) G.20 -~ seropositive (1 yr)
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- 29 - 2~
9 CEM) 0.60 infected cell line
10 CONT) 1.00 plasmid clone of HIV-I
11 CONT) 0.06 yeast DNA
12 CONT) 0.05 no DNA
13 NEG) 0.07 seronegative
14 NEG~ 0.06 seronegative
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SEQUENCE LISTINGr
( 1 ) GENERAL INFORMATION:
(i) APPLICANTS: Nectarios Tavernarakis,
George Hatzidakis, and
. Elias ~xambovitis
( ii) TITLE OF I~ENTION: RAPID AMPLIFICATION
AND DETECTION OF
NUCLEIC ACIDS
(iii) NUMBER OF SEQUENCES: S
(iv) CORRESPONDENCE ADDRESS:
(A) A~DRESSEE: Dickstein, Shapiro &
~orin
(B) STREET: 2101 L Street, N.W.
(C) CITY: Washington
(D) STATE: D.C.
(E) ~OUNTRY: U.S.A.
(F) ZIP: 2003~
(v) COMPUTER READABLE FORM:
~A) MEDIUM TYPE- Floppy disk
(B) COMPUTER: IB~ PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII
:
,
:,~
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- 31 - 2~75~83
(vi) CURRENT APPLICATION DATA:
(A) APPLICArrION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION: -
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) ~ILING DATE
~; (viii) ATTORNEY/AGENT INFORMATIOM:
, (A) NAME: Karny, Geoffrey M.
(B) REGISTRATION NUMBER: 31,382
(C~ ~OCKET NUMBER: I2277.001/P001
~,
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 785-9700
(B) T~LEFAX: (202~ ~87-0689
(2) INFORMATION FOR SEQ ID NO:I:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTHi 19 nucleotides
(B) TYPE: nucleic acid
~;: (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPrION: SEQ ID NO:l:
v
~ GAAGGAGCCA CCCCACAAG 19
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- 32 - 2 ~ 7'.~ 8~J3
.~ .
(3) INFORMATION FOR SEQ I~ NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 19 nucleotides :
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single :
(D~ TOPOLOGY: linear
~ii) MOLECULE TYPE: DNA
'~ , .
-~ (xi) SEQUENCE DESCRIPTION; SEQ ID NO:2:
'
~:~ CCCCCCTGTA GTTCGTCGG 19
'
(4~ INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
: (A) LENGTH: 24 nucleotides
(B~ TYPE: ~nucleic acid
(C) STRANDEDNESS- single
(D) TOPOLOGY: lin~ar
~ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO~3:
TTTAAACACC ATGCTAAACA CAGT 24
(5~ INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 nucleotides
(B) TYPE: nucleic acid
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- 33 -
(C) STRANDEDNESS: single
. (D) TOPOLOGY: linear
~,
, (ii) MOLECULAR TYPE: DNA
,
(xi) S~QUENCE DESCRIPTION: SEQ ID NO:4:
. GACATCGAGC TTGCTAGAAG 20
.
` (6) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 nucleotides
~,~! (B) TYPE: nucleic acid
(C) STRAND~DNESS: single
(D) TOPOLOGY linear
~` (ii) MOLECULE TYPE: DNA
: :
~- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GGTGACGAAT TCGGAGTTAT 20
(7) INFORMATION FOR SEQ ID NO:6:
` (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 nucleotides
~ (B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
.
:: : ~, .: , ,,

- 34 - 2~ 3
; :
: txi) SEQUENCE DESCRIPTION: SEQ ID NO:6: :
CTCTCTTTAA ACACCATGCT AACACAGT 28
~'`
'
:. :
-
.
,