Note: Descriptions are shown in the official language in which they were submitted.
1279~4
DETECTION OF VIRUSES BY AMPLIFICATION
AND HYBRIDIZATION
The present invention relates to a process for detecting the
presence or absence of a conserved, identifying nucleotide sequence of
_ 5 a virus. This invention also relates to a kit for such detection
having primers and a labeled hybridization probe.
The acquired immune deficiency syndrome (AIDS) is a
transmissible disorder of the cellular immune system resulting in
frequently fatal opportunistic infections or neoplasms. In addition,
10 AIDS is frequently complicated by central nervous system
dysfunction. The aetiologic agent(s) responsible for this disease has
been identified as a human retrovirus and designated as human T cell
leukemia virus III (HTLVIII), lymphadenopathy associated virus (LAV or
LAVA), and AIDS-associated virus (ARV-2). More recently, these
viruses have been collectively referred to as human immunodeficiency
virus (HIV). The isolates from the various lahoratories represent
identical or closely related viruses by numerous criteria (i.e.,
morphology, immunological cross-reactivities of envelope and
nucleocapsid proteins, nucleotide sequence, and entry into helper T
20 cells using the T4 antigen). A simian virus isolated from chimpanzees
and macaques suffering from symptoms indistinguishable from AIDS in
humans is also closely related by these same criteria. P. J. Kanki et
al., Science~ 230:951-954 (19~5).
One of the more intriguing observations about the viruses
associated with AIDS is their resemblance to the mature virion of
subfamily Lentiviridae. Members of this pathogenic but non-oncogenic
viral group include visna virus, and equine infectious anemia virus.
The similarities between the AIDS-associated viruses and lentiviruses
include virion morphology, immunological cross-reactivity, nucleotide
30 sequence, brain localization, replication, and marked heterogeneity.
The current immunodiagnostic tests to identify sera with
antibodies to the AIDS-associated virus(es) (see U.S. Patent No.
4,520,113 to Gdl10 et al.) are being used in blood banks to eliminate
potentially infectious blood. See also WO ~6/~1~34 published Mdrch
1~7~324~
27, 1986 (University of California) for retroviral polypeptides useful
in preparing monoclonal antibodies to detect retroviruses in the HTLV
family. ~ecause the similarities of the AIDS-associated viruses and
lentiviruses, in general, or visna specifically, may extend to the
ability of the virus(es) to reside as a DNA copy without producing
significant quantities of viral particles, a direct immunological
approach to detect AIDS-associated viruses may prove unsuccessful in a
significant fraction of persistently infected asymptomatic
individuals. Because the number of virus particles in the infected
tissues and blood may be few (due to viral quiescence), direct
detection of vira1 particles or RNA/DNA may be difficult, if not
impossible, without co-culturing the infected cells with a permissive
T cell line. Even with co-cultivation, the number of individuals
infected by HIV as indicated by virus isolation is an underestimate of
the true number of infected individuals; virus from only 50% ~IDS
patients, 85% of ARC, and 30% of healthy individuals at risk for AIDS
was isolated (Salahuddin et al., PNAS USA, ~ 5530-4 (1985)).
Saiki et al., Science, ~ 1350-1354 (19~5) describes a
process for amplifying nucleic acid sequences to facilitate detection
20 thereof, as by using a labeled RNA or DNA hybridization probe. In
this process primers are used to obtain primer extension products
which are used as templates to synthesize additional complementary
strands in the presence of nucleotides. The Saiki et al. article also
describes a technique whereby after a probe is hybridized to the
25 desired sequence, a restriction enzyme is added to cleave the hybrid
at a site within the desired sequence, and the restriction digest is
then analyzed for labeled fragments. Saiki et al., Biotechnology,
3.1008-1012 (1985) describe this latter technique in greater detail.
A review article by Landry et al., Clin. Lab. Med. (1985) ~
30 513-529 describes the field of nucleic acid hybridization as applied
to virus detection. W086/01535 published March 13, 1986 and EP
173,529 published March S, 1986 disclose molecular cloning of HTLVIII
and use of the clone as a probe to detect AIDS. Further, EP patent
publication 173,339, published March 5, 1986, discloses a genetic
35 analysis using a DNA probe to detect infections by foreign microbes.
~7~2~L
EP 185,444, published June 2~, 1986, discloses a recombinant peptide
for use as a probe to detect the HTLVIII virus in cell lysates. Oncor
Inc. announced in September, 1986 that it has developed a radioactive
blood test to detect the AIDS virus. U.S. Patent No. 4,591,552
discloses use of a labeled peptide to detect the presence of an
- antigen in a sample, particularly hepatitis B.
Use of a hybridization probe to detect latent viruses such
as AIDS and other viruses may allow identification of those
individuals who are persistently infected but are not producing virus
10 or individuals who are antibody negative but culture positive, and to
detect infected cells without the need to culture the virus.
Increasing the viral nucleic acid copy number of the virus by
amplification will facilitate the identification of viral nucleic acid
in infected individuals.
The present invention involves a process for detecting or
monitoring for the presence or absence of a nucleic acid sequence
which is substantially conserved among the nucleic acids in a virus
and specific to the nucleic acids in such virus and which nucleic acid
sequence is suspected of being contained in a sample, which process
20 comprises:
(a) treating the sample, together or separately, with an
oligonucleotide primer for each strand of the nucleic acid sequence,
four different nucleoside triphosphates, and an agent for
polymerization, under hybridizing conditions, such that for each
strand of the nucleic acid sequence an extension product of each
primer is synthesized which is complementary to each nucleic acid
strand, wherein said primer(s) are substantially complementary to each
strand of the nucleic acid sequence being detected or monitored, such
that the extension product synthesized from one primer, when it is
30 separated from its complement, can serve as a template for synthesis
of the extension product of the other primer;
(b) treating the samp1e under denaturing conditions;
(c) treating the product of step (b) with oligonucleotide
primers such that a primer extension product is synthesized using each
1;~79244
of the single strands produced in step (b) as a template, resulting in
amplification of the specific nucleic acid sequence or sequences if
present; and
(d) determining if the sequence to be detected is present
5 in the sample.
Preferably the sequence is in HIV (AIDS) and hepatitis B
viruses.
One way to detect the product is by adding to the product of
step (c) a labeled probe capable of hybridizing with the amplified
10 nucleic acid sequence; and determining whether the probe ~as
hybridized to an amplified sequence in the nucleic acid sample. In
one embodiment, this determination can be made by:
(1) digesting the hybridized mixture with a restriction
enzyme recognizing a site within the sequences in the probe; and
(2) detecting whether the restriction digest contains a
restriction fragment correlated with the presence cf the virus
sequence to be detected.
Before step (a) the nucleic acids in a patient sample may be
extracted therefrom so that the sample being treated is actually a
20 mixture of the extracted nuc1eic acids. In addition, the sample being
treated in step (a) need not be subjected beforehand to a process
wherein the virus in the sample is cultured.
In another embodiment, the invention herein relates to a kit
for detecting or monitoring for the presence or absence of a nucleic
acid sequence which is substantially conserved among the nucleic acids
in a virus and specific to the nucleic acids in a virus and which
nucleic acid sequence is suspected of being contained in a sample,
which kit comprises:
(a) one oligonuc1eotide primer for each strand of the
nucleic acid sequence to be detected, which primer or primers are
substantially complementary to each strand of each specific nucleic
acid seqùence such that an extension product synthesized from one
primer, when it is separated from its complement, can serve as a
12'-~24~
template for the synthesis of the extension product of the other
primer; and
(b) a probe capable of hybridizing with the nucleic
acid sequence.
_ 5 Preferably, the kit also contains an agent for
polymerization, four different nucleotides, and a means for detecting
hybrids of the probe and sequence.
The test kit herein may be used in research tests, clinical
tests and other diagnostic applications. In addition, it can be used
10 to detect infected cells without culturing the virus, a feature useful
in monitoring patients treated with various therapeutic agents to
resolve the infection.
Figure 1 is a schematic representation of the entire AIDS
genome, which consists of the long terminal repeat (LTR) noncoding
regions and the gag, pol, env, Q (or sor) and F (or 3'0RF) coding
regions, and shows from what region the primers were chosen for the
examples herein.
Figure 2 is a schematic representation of ten
oligonucleotides of the AIDS sequence which were ligated together to
form a 180 bp DNA fragment for a hybridization probe to be used in dot
blots to detect amplified products.
Figure 3 is a schematic flow diagram of how two clones,
M13mplOW:C7 and M13mplOW:D6, which contain nucleotide seqùence
alterations, were treated to obtain a sequence identical to the AIDS
virus. The resulting clone, M-13-GAG, contains the 180 bp insert for
use as a hybridization probe in dot blots. In the figure, X denotes
mutation in the gene.
The term Uvirus'' as used herein refers to any isolate or
series of related isolates that have a sequence that is substantially
conserved among and specific to the nucleic acids in the virus(es).
Examples of such viruses include HIY, hepatitis 8 virus, herpes
viruses, hepatitis A virus. rhinovirus, papilloma virus, Epstein-~arr
virus, etc. Preferred viruses herein are AIDS and hepatitis 8.
1279Z44
The present invention relates to a process and kit for
detecting or monitoring for a nucleic acid sequence associated with d
virus in a sample of nucleic acid(s) suspected of containing the
sequence. The following discussion relates to the HIV and hepatitis B
virus in particular, but could be applied to any virus as defined
- herein.
Because HIV is highly variable, there is a need to find a
common denominator (conserved region with a length which allows a
specific primer as defined herein to initiate polymerization) among
10 the variants of the virus to use for detecting a significant fraction
of the viruses associated with AIDS. A significant fraction is that
number of individuals sufficient to make the test diagnostically or
commercially feasible. Currently four HIVs, ARV, HTLVIII, LAV and
LAVA, have been se~uenced, and five human hepatitis B viruses as well
15 as related viruses that infect woodchucks, ground squirrels and ducks,
have been sequenced. The initial four HIVs and their associated
variants are designated herein as "AIOS viruses," and the five
hepatitis B viruses, and the hepatitis B-related viruses that infect
woodchucks, ground squirrels and ducks, are designated herein as
"hepadnaviruses." The sequence to be amplified also must be specific
to the AIDS viruses or to hepadnaviruses, i.e., not react with HTLVI
or HTLVII or other non~AIDS viruses, or not react with non-
hepadnaviruses, respectively.
The entire genome of the four HIV isolates and their
25 variants is provided by Sanchez-Pescador et al., Science, ?27, 484-492
(1985) for AR~; Starcich et al., Science, 227, 538-540 (1985) for
HTLVIII; Wain-Hobson et al., Cell, ~1 9-17 (1985) for LAV; and
Muesing et al., Nature, 313, 450-458 (1985) for LAVA. There is a
general consensus that these viruses are all variants of the same
30 strain. They are completely conserved within a species, i.e., they
have no variants.
The entire genome of the duck hepatitis B virus is provided
in J. Virol., 49:782-792 (1984), and the human and woodchuck virus
genomes are provided in the references cited in the J. Virol. paper.
1~79~4~
The entire genome of the ground squirrel hepatitis B virus is provided
in J. Virol., 41:51-65 (1982).
The term "substantially conserved" as applied to the
sequence to be detected signifies that the sequence must be
sufficiently complementary to the nucleic acids in the virus being
detected to initiate polymerization at least at room temperature in
the presence of an agent for polymerization and the four nucleoside
triphosphates.
The primers used will be oligonucleotides of any length and
10 sequence so as to provide specific initiation of polymerization on a
significant number of nucleic acids in the virus. Specifically, the
term "primer" as used herein refers to a molecule comprised of two or
more deoxyribonucleotides or ribonucleotides, preferably more than
three, which is capable of acting as a point of initiation of
synthesis when placed under conditions in which synthesis of a primer
extension product which is substantially complementary to a nucleic
acid strand is induced, i.e., in the presence of nucleoside
triphosphates and an agent for polymerization such as DNA polymerase
and at a suitable temperature and pH. The primer is preferably single
stranded for maximum efficiency in amplification, but may
alternatively be double stranded. If double stranded, the primer is
first treated to separate its strands before be;ng used to prepare
extension products. Preferahly, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent for polymerization. The exact lengths of the primers
will depend on many factors, including temperature, buffer, nucleotide
composition and source of primer. For purposes herein, the
oligonucleotide primer typically contains 15-2~ or more nucleotides,
although it may contain fewer nucleotides. If the virus constitutes
the AIDS viruses, preferably the primers are from the gag region. If
the virus constitutes the hepadnaviruses, preferably the primers are
from the polymerase or envelope genes of those viruses.
1 2 7~ L
The primers herein are selected to be "substantially"
comp1ementary to each strand of the specific sequence to be
amplified. This means that the primers must be sufficiently
complementary to hybridize with their respective strands under
conditions which allow the agent for polymerization to perform, i.e.,
- the primers have sufficient complementarity with the sequence of the
strand to be amplified to hybridize therewith and thereby form a
template for synthesis of the extension product of the other primer.
Preferably, the primers have exact complementarity with the strand.
One may select the sequence being amplified from among the
regions that are substantially conserved among the related viruses of
interest. Therefore, the primers and probes may be identified by any
suitable means. This may be done manually by comparing the regions of
the published nucleic acid sequences of the relevant viral genomes,
e.g., the four AIDS viral genomes and the four hepatitis B genomes.
Another more convenient method is to use a computer program
to compare the sequences. For this purpose, a commercial program with
the underlying computer algorithm supplied by National Biomedical
Research Foundation using a dot matrix may be conveniently employed.
This program involves inputting the nucleic acid sequences of the
various related vlruses of interest and defining a window size for
base pair homology. The program employs graphics to compare the
sequences on different axes, and a dot appears where there is at least
substantial homology. Preferably, the window size is greater than six
bases.
For the AIDS viruses, a dot matrix program reveals that the
gag region of the genome (see Figure 1), also known as the
nucleocapsid gene, is most conserved among the coding regions in the
four variants. The next most conserved coding region is the pol
regior,, followed by the env region of the genome. ~ecause gag is most
conserved among the coding regions, it is the preferred region from
which to select primers and probes for detecting the sequence.
Regions of the viral genome that do not encode proteins can
also be used to determine a sequence for the primers to be used. For
~;~79244
purposes herein, to maximize sensitivity and specificity, the sequence
being detected is homologous with a sequence of a length sufficient to
allow specific priming which is substantially conserved among the
related viruses, particularly at the restriction cleavage site if a
probe and restriction enzyme are employed.
The techniques used for amplifying and thereafter detecting
the product are described in detail in Saiki et al., Biotechnolo~y,
supra and Saiki et al., Science, supra. In general, the amplification
process involves an ènzy~atic chain reaction for preparing, in
exponential quantities relative to the number of reaction steps
involved, a specific nucleic acid sequence, given that the ends of the
required sequence are known in sufficient detail that oligonucleotide
primers can be synthesized which will hybridize to them, and that a
small amount of the sequence is available to initiate the chain
reaction. One primer is co~4lementary to the negative (-) strand and
the other is complementary to the positive (+) strand. Annealing the
primers to denatured nucleic acid followed by extension with an enzyme
such as the large fragment of DNA Polymerase I (Klenow) and
nucleotides results in newly synthesized + and - strands containing
the target sequence. Because these newly synthesized sequences are
also templates for the primers, repeated cycles of denaturing,, primer
annealing and extension results in exponential accumulation of the
region defined by the primer. The product of the chain reaction will
be a discrete nucleic acid duplex with termini corresponding to the
ends of the specific primers employed.
The amplification process is illustrated diagrammatically
below, where double-stranded DNA containing the desired sequence ES]
comprised of complementary strands [S+~ and [S~] is utilized as the
nucleic acid. During the first and each subsequent reaction cycle
extension of each oligonucleotide primer on the original template will
produce one new ssDNA molecule product of indefinite length which
terminates with only one of the primers. These products, hereafter
referred to as "long products," will accumulate in a linear fashion;
that is, the amount present after any number of cycles will be
proportional to the number of cycles.
~X7~4~
The long products thus produced will act as templates for
one or the other of the oligonucleotide primers during subsequent
cycles and will produce molecules of the desired sequence [S~] or [S ]
These molecules will also function as templates for one or the other
5 of the oligonucleotide primers, producing further [S+] and ~S ], and
_ thus a chain reaction can be sustained which will result in the
accumulation of [S] at an exponential rate relative to the number of
cycles.
By-products formed by oligonucleotide hybridizations other
10 than those intended are not self-catalytic and thus accumulate at a
linear rate.
The specific sequence to be amplified, [S], can be depicted
diayrammatically as:
[S+] S' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCC 3'
15 [S~] 3' TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5'
The appropriate oligonucleotide primers would be:
Primer 1: 3' GGGGGGGGGG 5'
Primer 2: 5' AAAAAAAAAA 3'
so that if DNA containing [S]
20 ....zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz....
....zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz....
is separated into single strands and its single strands are hybridized
to Primers 1 and 2, the following extension reactions can be catalyzed
by DNA polymerase in the presence of the four deoxyribonucleoside
25 triphosphates:
3' 5'
extends~ GGGGGGGGGG Primer 1
....zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz....
original template strand+
30 original template strand~
....zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz....
127~44
11
Primer 2 AAAAAAAAAA )extends
5' 3'
On denaturation of the two duplexes formed, the products are:
_ 3' 5'
....zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG
newly synthesized long product 1
....zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXX XCCCCCCCCCCzzzzzzzzzzzzzzzz....
original template strand+
3~ 5,
....zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz....
original template strand~
5' 3'
AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz....
newly synthesized long product 2
If these four strands are allowed to rehybridize with Primers 1 and 2
in the next cycle, the agent for polymerization will catalyze the
following reactions:
Primer 2 5' AAAAAAAAAA - )extends to here
3'....zzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5'
newly synthesized long product l
extends~ GGGGGGGGGG S' Primer 1
5'....zzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzz....3'
original template strand+
Primer 2 5' A M AAAAAAA ~ ) extends
3'....zzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYGGGGGGGGGGzzzzzzzzzz....5'
original template strand~
extends to here ~ GGGGGGGGGG 5' Primer 1
1279Z4~L
5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz..3'
newly synthesized long product 2
If the strands of the above four duplexes are separated, the following
strands are found:
5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCC 3'
_ newly synthesized ~S+]
3'....zzzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5'
first cycle synthesized long product 1
3'....zzzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5'
10 newly synthesized long product 1
S'....zzzzzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzz....3'
original template strand+
5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz...3'
newly synthesized long product 2
3'..zzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz...5'
original template strand~
- 3' TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5'
newly synthesized [S~]
5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzz...3'
first cycle synthesized long product 2
It is seen that each strand which terminates with the
oligonucleotide sequence of one primer and the complementary sequence
of the other is the specific nucleic acid sequence [S] that is desired
to be produced.
The steps of this process can be repeated indefinitely,
being limited only by the amount of Primers 1 and 2, inducing agent
and nucleotides present. The amount of original nucleic acid remains
constant in the entire process, because it is not replicated. The
amount of the 1ong products increases linearly because they are
30 produced only from the original nucleic acid. The amount of the
specific sequence increases exponentially. Thus, the specific
sequence will become the predominant species. This is illustrated in
the following table, which indicates the relative amounts of the
species theoretically present after n cycles, assuming 100~ efficiency
35 at each cycle:
12>7~Z4~
Number of Double Strands
After 0 to n Cycles
Long Specific
Cycle Number Template ProductsSequence [S]
0 1 - -
0
2 1 2
3 1 3 4
1 5 26
10 10 1 10 1013
1 15 32,752
1 20 1,048,555
n 1 n (2n-n-1)
When a single-stranded nucleic acid is utilized as the template, only
15 one long product is formed per cycle.
As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes of which cut double-
stranded DNA at or near a specific nucleotide sequence.
The primer(s) herein may be selected by the following
20 criteria, which are factors to be considered, but are not exclusive or
determinative. First, the primers are selected from conserved regions
of the viral genome. In the case of the AIDS genome, the gag region
(nucleocapsid gene) is the most conserved of the coding regions,
followed by the pol and env regions, and therefore, the gag region was
25 chosen for initial studies. In the case of the hepatitis B genome,
the entire coding region of the genome is conserved within a single
species.
Secondly, the primer 1acks homology with any sequences of
viral genomes that would be expected to compromise the test. For
30 example, those sequences for HTLVI, published by Seiki, M. et al.,
PNAS (USA) 80:3618-3622 (1983), would compromise the test for AIDS.
~79~4a~
Third, the primer preferably lacks secondary structure
formation in the amplified nucleic acid which may interfere with
extension by the amplification enzyme such as E. coli DNA polymerase,
preferably that portion of the DNA polymerase referred to as the
Klenow fragment. This may be accomplished by employing up to about
- 15% by weight, preferably 5-10% by weight, dimethyl sulfoxide (DMSO)
in the amplification medium and/or increasing the amplification
temperatures to 30-40C, preferably 35-40~C.
Fourth, the primer preferably has an approximate 50~ content
10 of guanine and cytosine, and does not contain multiple consecutive
adenine and thymine residues at the 3' end of the primer which may
result in less stable hybrids.
Finally, if the amplified product will be detected by use of
a restriction enzyme, the probe must have an internal lnon-terminal)
restriction site.
The oligonucleotide primers may be prepared using any
suitable method, such as, for example, the phosphotriester and
phosphodiester methods described above, or automated embodiments
thereof. In one such automated embodiment diethylphosphoramidites are
20 used as starting materials and may be synthesized as described by
Beaucage et al., Tetrahedron Letters (1981), 22:1859-1862. One method
for synthesizing oligonucleotides on a modified solid support is
described in U.S, Patent No. 4,458,066. It is also possible to use a
primer which has been isolated from a biological source (such as a
25 restriction endonuclease digest).
Any source of nucleic acid, in purified or nonpurified form,
can be utilized as the starting nucleic acid or acids, provided it
contains or is suspected of containing the specific nucleic acid
sequence associated with the virus to be detected. Thus, the process
30 may employ, for example, DNA or RNA, including messenger RNA, which
DNA or RNA may be single stranded or double stranded. In the event
that RNA is to be used as a template, enzymes and/or conditions
optimal for reverse transcribing the template to DNA would be
utilized. In addition, a DNA-RNA hybrid which contains one strand of
~z7924~1
each may be utilized. A mixture of any of these nucleic acids may
also be employed, or the nucleic acids produced from a previous
amplification reaction herein using the same or different primers may
be so utilized. The specific nucleic acid sequence to be amplified
may be only a fraction of a larger molecule or can be present
_ initially as a discrete molecule, so that the specific sequence
constitutes the entire nucleic acid. It is not necessary that the
sequence to be amplified be present initially in a pure form; it may
be a minor fraction of a complex mixture, such as a portion of the
virus-encoding gene contained in whole human DNA. The starting nucleic
acid may contain more than one desired specific nucleic acid sequence
which may be the same or different. Therefore, the present process is
useful not only for producing large amounts of one specific nucleic
acid sequence, but also for amplifying simultaneously more than one
different specific nucleic acid sequence located on the same or
different nucleic acid molecu1es,
The nucleic acid(s) may be obtained from any source, for
examp1e, natural DNA or RNA from higher organisms such as animals.
DNA or RNA may be extracted from a bodily sample such as blood, tissue
20 material such as chorionic villi, or amniotic cells by a variety of
techniques such as that described hy ~aniatis et al., ~olecular
Cloning (1982), 280-281.
If the sample is impure such as plasma, seru~ or blood,
before amplification it may be treated with an amount of a reagent
effective to open the cells, fluids, tissues, viral capsids or animal
cell membranes of the sample, and to expose and/or separate the
strand(s) of the nucleic acid(s). This lysing and nucleic acid
denaturing step to expose and separate the strands will allow
amplification to occur much more readily. In addition, the AIDS virus
30 need not be cultivated in the sample before the sample is treated with
the amplification reagents. The sample may be centrifuged to obtain
buffy coats, which are then passed through a co1umn to obtain
leukocytes. The leukocytes may then be treated to extract the nucleic
acids therefrom for use as the sample to be amplified.
lZ79~4~
Any specific nucleic acid sequence can be produced by the
present process. It is only necessary that a sufficient number of
bases at both ends of the sequence be known in sufficient detail so
that two oligonucleotide primers can be prepared which will hybridize
5 to different strands of the desired sequence and at relative positions
- along the sequence such that an extension product synthesized from one
primer, when it is separated from its template (complement), can serve
as a template for extension of the other primer into a nucleic acid of
defined length. The greater the knowledge about the bases at both
10 ends of the sequence, the greater can be the specificity of the
primers for the target nucleic acid sequence, and thus the greater the
efficiency of the process. It will be understood that the word primer
as used hereinafter may refer to more than one primer, particularly in
the case where there is some ambiguity in the information regarding
15 the terminal sequence(s) of the fragment to be amplified. For
instance, in the case where a nucleic acid sequence is inferred from
protein sequence information a collection of primers containing
sequences representing all possible codon variations based on
degeneracy of the genetic code will be used for each strand. One
20 primer from this collection will be substantially conserved with the
end of the desired sequence to be amplified.
The specific nucleic acid sequence is produced by using the
nucleic acid containing that sequence as a template. If the target
nucleic acid sequence of the sample contains two strands, it is
necessary to separate the strands of the nucleic acid before it can be
used as the template, either as a separate step or simultaneously with
the synthesis of the primer extension products. This strand
separation can be accomplished using any suitable denaturing
conditions, including physical, chemical or enzymatic means, the word
30 "denaturing" used herein to include all such means. One physical
method of separating the strands of the nucleic acid involves heating
the nucleic acid until it is denatured. Typical heat denaturation may
involve temperatures ranging from about 80 to 105C for times ranging
from about 1 to 10 minutes. Strand separation may also be induced by
35 an enzyme from the class of enzymes known as helicases or the enzyme
~27~44
17
RecA, which has helicase activity and in the presence of riboATP is
known to denature DNA. The reaction conditions suitable for
separating the strands of nucleic acids with helicases are described
by Kuhn Hoffmann-Ber1ing, CSH-Quantitative Biolo~y, 43:63 (1978), and
techniques for using RecA are reviewed in C. Radding, Ann. Rev.
_ ~ene~ 16:405-37 (1982).
If the original nucleic acid containing the sequence to be
amplified is single stranded, its complement is synthesized by addin~
one or two oligonucleotide primers thereto. If an appropriate single
I0 primer is added, a primer extension product is synthesized in the
presence of the primer, an agent for polymerization, and the four
nucleoside triphosphates described below. The product will be
partially complementary to the single-stranded nucleic acid and will
hybridize with the nucleic acid strand to form a duplex of unequal
length strands that may then be separated into single strands as
described above to produce two single separated complementary
strands. Alternatively, two appropriate primers may be added to the
single-stranded nucleic acid and the reaction carried out.
If the original nucleic acid constitutes the sequence to be
amplified, the primer extension product(s) produced will be completely
or substantially complementary to the strands of the original nucleic
acid and will hybridize therewith to form a duplex of equal length
strands to be separated into single-stranded molecules.
When the complementary strands of the nucleic acid or acids
25 are separated, whether the nucleic acid was originally double or
single stranded, the strands are ready to be used as a template for
the synthesis of additional nucleic acid strands. This synthesis is
performed under conditions allowing hybridization of primers to
templates to occur. Generally it occurs in a buffered aqueous
30 solution, preferably at a pH of 7-9, most preferably about 8.
Preferably, a molar excess (for genomic nucleic acid, usually about
108:1 primer:template) of the two oligonucleotide primers is added to
the buffer containing the separated template strands. It is
understood, however, that the amount of complementary strand may not
1279Z~4
be known if the process herein is used for diagnostic applications, so
that the amount of primer relative to the amount of complementary
strand cannot be determined with certainty. As a practical matter,
however, the amount of primer added will generally be in molar excess
5 over the amount of complementary strand (template) when the sequence
- to be amplified is contained in a mixture of complicated long-chain
nucleic acid strands. A large molar excess is preferred to improve
the efficiency of the process.
The deoxyribonucleoside triphosphates dATP, dCTP, dGTP and
10 TTP are also added to the synthesis mixture, either separately or
together with the primers, in adequate amounts and the resulting
solution is heated to about 90-100C for from about 1 to 10 minutes,
preferably from 1 to 4 minutes. After this heating period the
solution is allowed to cool to room temperature, which is preferable
for the primer hybridization. To the cooled mixture is added an
appropriate agent for effecting the primer extension reaction (called
herein "agent for polymerization"), and the reaction is allowed to
occur under conditions known in the art. The agent for polymerization
may also be added together with the other reagents if it is heat
20 stable. This synthesis reaction may occur at from room temperature up
to a temperature above which the agent for polymerization no longer
functions. Thus, for example, if DNA polymerase is used as the agent,
the temperature is generally no greater than about 40C. Most
conveniently the reaction occurs at room temperature.
The agent for polymerization may be any compound or system
which will function to accomplish the synthesis of primer extension
products, including enzymes. Suitable enzymes for this purpose
include, for example, E. coli DNA polymerase I, Klenow fragment of E.
coli DNA polymerase I, T4 DNA polymerase, other available DNA
30 polymerases, polymerase mutejnS, reverse transcriptase, and other
enzymes, including heat-stable enzymes (i.e., those enzymes which
perform primer extension after being subjected to temperatures
sufficiently elevated to çause denaturation), which will facilitate
combination of the nucleotides in the proper manner to form the primer
35 extension products which are complementary to each nucleic acid
~'~79244
19
strand. Generally, the synthesis will be initiated at the 3' end of
each primer and proceed in the 5' direction along the template strand,
until synthesis terminates, producing molecules of different
lengths. There may be agents for polymerization, however, which
initiate synthesis at the 5' end and proceed in the other direction,
- using the same process as described above.
The newly synthesized strand and its complementary nucleic
acid strand will form a double-stranded molecule under the hybridizing
conditions described above if the target sequence is present, and this
10 hybrid is used in the succeeding steps of the process. In the next
step, the sample treated under hybridizing conditions is subjected to
denaturing conditions using any of the procedures described above to
provide single-stranded molecules if the target sequence is present.
New nucleic acid is synthesized on the single-stranded
15 molecules. Additional agent for polymerization, nucleotides and
primers may be added if necessary for the reaction to proceed under
the conditions prescribed above. Again, the synthesis will be
initiated at one end of each of the oligonucleotide primers and will
proceed along the single strands of the template to produce additional
20 nucleic acid. After this step, half of the extension product will
consist of the specific nucleic acid sequence bounded by the two
primers.
The steps of denaturing and extension product synthesis can
be repeated as often as needed to amplify t~e target nucleic acid
sequence to the extent necessary for detection~ As will be described
in further detail below, the amount of the specific nucleic acid
sequence produced will accumulate in an exponential fashion,
When it is desired to produce more than one specific nucleic
acid sequence from the first nucleic acid or mixture of nucleic acids,
30 the appropriate number of different oligonucleotide primers are
utilized. For example, if two different specific nucleic acid
sequences are to be produced, four primers are utilized. Two of the
primers are specific for one of the specific nucleic acid sequences
and the other two primers are specific for the second specific nucleic
X4fl
acid sequence. In this manner, each of the two different specific
sequences can be produced exponentially by the present process.
The present invention can be performed in a step-wise
fashion where after each step new reagents are added, or
5 simu1taneously, where all reagents are added at the initial step, or
partially step-wise and partially simultaneous, where fresh reagent is
added after a given number of steps. If a method of denaturation,
such as heat, is employed which will inactivate the agent for
polymerization, as in the case of a heat-labile enzyme, then it is
Io necessary to replenish the agent after every strand separation step.
The simultaneous method may be utilized when an enzymatic means is
used for the strand separation step. In the simultaneous procedure,
the reaction mixture may contain, in addition to the nucleic acid
strand(s) containing the desired sequence, the strand-separating
15 enzyme (e.g., helicase), an appropriate energy source for the strand-
separating enzyme, such as rATP, the four nucleoside triphosphates,
the oligonucleotide primers in molar excess, and the agent for
polymerization, e.g., Klenow fragment of E. coli DNA polymerase I.
If heat is used for denaturation in a simultaneous process,
20 a heat-stable agent such as a thermostable polymerase may be employed
which will operate at an elevated temperature, preferably 50-105C
depending on the asent, at which temperature the nucleic acid will
consist of single and double strands in equilibrium. For smaller
lengths of nucleic acid, lower temperatures of about 40-50C may be
employed. The upper temperature will depend on the temperature at
which the enzyme will degrade or the temperature above which an
insufficient level of primer hybridization will occur. Such a heat-
stable enzyme is described, e.g., by A. S. ~aledin et al., 8iokhimiya,
45, 644-651 (1980). For this constant temperature reaction to
30 succeed, the primers have their 3' ends within 6-8 base pairs of each
other. Each step of the process will occur sequentially
notwithstanding the initial presence of all the reagents. Additional
materials may be added as necessary. After the appropriate length of
time has passed to produce the desired amount of the specific nucleic
acid sequence, the reaction may be halted by inactivating the enzymes
in any known manner or separating the components of the reaction.
~7~4
The amplification may also be carried out using a
temperature-cycling reaction wherein the temperature is continually
increased to 90-105C to allow for denaturation and then decreased to
35-65C or more to allow for extension and annealing using a heat-
5 stable enzyme. In practical terms the temperature simply raised to_ about 95~C, lowered to about 65C or to as low as 37~C and raised
again to about 95C, and the cycle is continued for the desired
period.
In another embodiment of an automated process, the process
IO of the present invention may be conducted continuously by cycling the
reaction through a denaturing region, a reagent addition region, and a
reaction region. In another embodiment, the enz~ne used for the
synthesis of primer extension products can be immobilized in a column.
The other reaction components can be continuously circulated by a pump
15 through the column and a heating coil in series, thus the nucleic
acids produced can be repeatedly denatured without inactivating the
enzyme.
The amplified product may be detected by analyzing it by
Southern blots without using radioactive probes. In such a process,
20 for example, a small sample of DNA from, e.g., peripheral blood
lymphocytes containing a very low leve1 of the sequence associated
with, e.g., AIDS, is amplified, and analyzed via a Southern blotting
technique. The use of non-radioactive probes is facilitated by the
high level of the amplified signal.
Another method of detection involves detection using a
labeled probe capable of hybridizing with the amplified nucleic acid
sequence and determining if the probe has hybridized. Such probe
necessarily contains a substantially conserved nucleic acid sequence
from the genome of the virus (for the AIDS virus, HTLVIII, ARV, LAV,
30 LAVA, or a variant thereof) and is selected as described above for
primers and amplified sequences. For AIDS, preferably the probe is
selected from the gag region of the AIDS genome.
One such probe method involves the oligomer restriction
technique described in EP Patent Publication No. 164,054 published
35 December 11, 1985. In this procedure, the a~plified nucleic acid is
~2'79Z4a~
denatured and hybridized in solution to a labeled oligonucleotide
probe which hybridizes specifically to the target sequence ~spans the
particular conserved region containen by the primers) and spans at
least one restriction site of interest. The duplex formed between
5 target and probe will reconstitute the restriction site, and when
- cleaved with restriction enzyme, such as, e.g., BstNI, PvuII, DdeI, or
Dral, releases a labeled probe fragment which can be resolved from the
full-length probe by gel electrophoresis. The resulting gel is then
autoradiographed. Analysis of the amplified product by this method is
10 rapid, i,e., results c~n be obtained in a few hours. Preferably, the
probe is 30-50 bases long and is labeled. Also, preferably the
restriction enzyme is BstNI or PvuII if the sequence to be detected is
from the AIDS virus and DdeI if the sequence to be detected is from
the hepadnavirus.
Another method which may be used to analyze the amplified
product is the dot blot method. In this method, the amplified samples
are spotted directly on a membrane and hybridized with a labeled
probe. The label may be detected by spectroscopy, photochemistry or
by biochemical, immunochemical or chemical means. Examples include
20 enzymes such as alkaline phosphatase, a radioactive label such as 32p,
a fluorescent label, or biotin. In one embodiment, the probe is a
biotinylated probe in which the biotin is attached to a spacer arm of
the formula:
H
(CH2)2 [(cH2)xo]y-cH2cH2-N-
where Y is 0, NH or N-CH0, x is a number from 1 to 4, and y is a
number from 2 to 4. The spacer arm is in turn attached to a psoralen
moiety of the formula:
CH3 CH2-
CH3
CH3
7~3Xq~
The psoralen moiety intercalates into and crosslinks a "gapped circle"
probe as described by Courage-Tebbe et al., Biochim. Biophys. ~cta,
697 (1982) 1-5, wherein the single-stranded hybridization region of
the gapped circle spans the region contained between the primers. The
5 details of this biotinylation and dot blot procedure are described
- more fully in commonly assigned U.S. Patent Nos. 4,582,789 and
4,617,261. The biotinylated probes eliminate the need for radioactive
isotopes.
Alternatively, the probe may be spotted on the membrane
10 first under prehybridization conditions if necessary and then the
amplified product is added to the pre-treated membrane under
hybridization conditions, "in a reverse" dot blot format.
The dot blot procedure is more time-consumin~ than the
oligomer restriction method described above, because the membrane mlst
first be prehybridized and then hybridized with the probe. However,
with rapidly mutating viruses, it has the advantage that sequences
containing limited base mismatches are still detected under
wppropriate hybridizing conditions, whereas with the oligomer
restriction method, any virus harboring a mutation which results in
20 the abolishment of the restriction site will not be detected due to
the variability of the virus.
The invention herein also contemplates a kit format which
comprises a packaged multicontainer unit having containers of each
primer and the probe utilized. The kit may also have a container with
25 the agent for polymerization to synthesize the primer extension
products, such as enzymes, a container with each of the four
nucleoside triphosphates, and a container with means to detect the
label tsuch as an avidin-enzyme complex if the label is biotin). In
addition, the kit may have a container which includes a positive
control containing one or more nucleic acids with a sequence of the
viral genome of interest (e.g., ~IDS) and/or a container including a
negative control without such nucleic acids. Moreover, the kit may
have a container for each restriction enzyme capable of cleaving a
nucleic acid containing the target sequence at a site contained in a
sequence in the probe.
~L27~2~
24
The following examples illustrate various embodiments of the
invention and are not intended to be limiting in any respect. In the
examples all parts and percentages are by weight if solid and by
volume if liquid and all temperatures are in degrees Centigrade,
5 unless otherwise indicated.
EXAMPLE 1
The desired sequences to be amplified were contained in
eleven coded DNA samples obtained from Dr. Bernard Poiesz of the
Reyional Oncology Center, SUNY Upstate Medical Center, Syracuse, New
10 York 13210, identified as 194BK, 342, 367, 361, 368H, 207, 307, 30 ~ ,
323, 326 and 340. The sequences to be amplified, the primers, and the
probes were identified by the dot matrix program as described above,
wherein the sequence window selected was at least 20 base pairs long,
so that the sequences were chosen within conserved regions of the AIDS
15 viruses.
The coded samples were first cultured in the presence of
interleukin-2 by Dr. Poeisz to test for the presence of virus. Then,
the DNA was extracted from the samples by t~e following procedure:
1. 1-2 x 108 cultured cells were lysed in tubes with 20 ml
20 of sodium dodecyl sulfate lysis buffer (1% SDS, 150 mM NaCl, 25 mM Na2
EDTA).
2. 400 ul of a 5 mg/~l solution of proteinase K was added
per tube and incubated at 37~C overnight.
3. The DNA was sequentially extracted with phenol and
25 CHCl3:isoamyl alcohol followed by precipitation with ethanol.
4. The DNA was spooled out on a glass rod and resuspended
in 1 x TE buffer (10 mM Tris, 1 ~M Na2EDTA, pH 7.5) and dialyzed
exhaustively against 1 x TE buffer.
30 1. Synthesis of Primers
The following two oligodeoxyribonucleotide primers,
designated SK01 and SK02, respectively, were prepared hy the method
described below:
5'-CAGGGAGCTAGAACGAT-3' (SK01)
5'-CTTCTGATCCTGTCTGA-3' (SK02 )
(SKOl and SK02 were selected to provide for amplification of 107 bases
between nucleotides 900 and 1006 of HTLVlII-isolate BH10.)
A. Automated Synthesis Procedures: The
diethylphosphoramidites, synthesized according to Beaucage and
Caruthers (Tetrahedron Letters (1981) 22:1859-1862) were sequentially
condensed to a nucleoside derivatized controlled pore glass support
using a Biosearch SAM-1. The procedure included detritylation with
10 trichloroacetic acid in dichloromethane, condensation using
benzotriazole as activating proton donor, and capping with acetic
anhydride and dimethylaminopyridine in tetrahydrofuran and pyridine.
Cycle time was approximately 30 minutes. Yields at each step were
essentially quantitative and were determined by collection and
15 spectroscopic examination of the dimethoxytrityl alcohol released
during detritylation.
B. Oligodeoxyribonucleotide Deprotection and Purification
Procedures: The solid support was removed from the column and exposed
to 1 ml concentrated ammonium hydroxide at room temperature for four
20 hours in a closed tube. The support was then removed by filtration
and the solution containing the partially protected
oligodeoxynucleotide was brought to 55C for five hours. Ammonia was
removed and the residue was applied to a preparative polyacrylamide
gel. Electrophoresis was carried out at 30 volts/cm for 90 minutes
25 after which the band containing the product was identified by UV
shadowing of a fluorescent plate. The band was excised and eluted
with 1 ml distilled water overnight at 4C. This solution was applied
to an Altech RP18 column and eluted with a 7-13% gradient of
acetonitrile in 1% ammonium acetate buffer at pH 6Ø The elution was
30 monitored by UV absorbance at 260 nm and the appropriate fraction
collected, quantitated by UV absorbance in a fixed volume and
evaporated to dryness at room temperature in a vacuum centrifuge.
C. Characterization of Oligodeoxyribonucleotides: Test
aliquots of the purified oli~onucleotides were 32p 1abeled with
17~7~24~
polynucleotide kinase and y-32P-ATP. The labeled compounds were
examined by autoradiography of 14-20~ polyacrylamide gels after
electrophoresis for 45 minutes at 50 volts/cm. This procedure
verifies the molecular weight. Base composition was determined by
5 digestion of the oligodeoxyribonucleotide to nucleosides by use of
- venom diesterase and bacterial alkaline phosphatase and subsequent
separation and quantitation of the derived nucleosides using a reverse
phase HPLC column and a 10~ acetonitrile, 1~ ammonium acetate mobile
phase.
10 Il. Amplification Reaction
One microgram of DNA from each of the eleven coded DNA
samples from Dr. Poiesz was added to 100 ~l of buffer consisting of 10
mM Tris-HCl, pH 7.5, 50 mM sodium chloride and 10 ~M magnesium
lS chloride and containing 100 picomoles of Primer SK01, 100 picomoles of
Primer SK02, and 150 nanomoles each of dATP, dCTP, dGTP and TTP.
The resulting solution was heated to 100C for 10 minutes
and allowed to cool to room temperature for two minutes, whereupon 2
~l containing one unit of Klenow fragment of E. coli DNA polymerase
20 was added. The reaction was allowed to proceed for two minutes at
room temperature, after which the enzyme was inactivated by heating at
95C fcr two minutes, The denaturation, primer annealing, and
extension with Klenow, two minutes per step, and adding polymerase
were repeated nineteen times.
25 III. Synthesis and Phosphor~lation of Oligodeoxyribonucleotide Probe
A labeled DNA probe, SK03, of the sequence:
5-*AATCCTGGCCTGTTAGAAACATCAGAAG-3',
where * indicates the label, was synthesized according to the
procedures described in Section I. The probe was labeled by
30 contacting 10 pmole thereof with 4 units of T4 polynucleotide kinase
and 50 pmole y 32P-ATP (about 7200 Ci/mmole) in a 40 ~l reaction
volume containing 70 mM Tris buffer (pH 7.6), 10 mM MgC12, 1.5 mM
spermine, and 2.5 mM dithiothreitol for 90 minutes at 37C. The total
~792~4
27
volume was then adjusted to 100 ~l with 25 mM EDTA and an aliquot
removed for determination of specific activity by TCA precipitation.
The labeled probe was concentrated using a vacuum apparatus and
purified by electrophoresis on a 18% polyacrylamide gel in Tris-boric
5 acid-EDTA (TBE) buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH
- 8.3) for 500 vhr. After localization by autoradiography, the portion
of the gel containing the labeled probe was excised, crushed and
eluted into 0.2 ml TE buffer overnight at 4C. TCA precipitation of
the reaction product indicated that the specific activity was 2
10 Citmmole and the final concentration was 20 pmole/ml.
IV. Hybridization/Digestion of Amplified Genomic DNA with Probe and
BstN1
Ten microliters of amplified DNA (containing the pre-
amplification equivalent of 71 ng of genomic DNA) was dispensed into a
15 1.5 ml Microfuge tube and 20 ~l of TE buffer to a final volume of 30
~l. The sample was denatured at 95C for 10 minutes. Ten microliters
of 0.6 M NaCl containing 0.02 pmole of SK03 probe was added to the
tube, mixed gently, overlayed with mineral oil, and immediately
transferred to a 56C heat block for one hour. Ten microliters of 50
20 mM MgC12 and 1 ~l of BstNI (10 units) were added and the reannealed
DNA was digested for 30 minutes at 56C. The reaction was stopped by
adding 4 ~l 75 mM EDTA and 6 ~l tracking dye to a final volume of 60
The mineral oil was extracted with 0.2 ml chloroform, and 13
25 ~l of the reaction mixture (~15 ng genomic DNA) was loaded onto a 30%
polyacrylamide mini-gel in an electrophoresing apparatus. The gel was
electrophoresed at approximately 300 volts for one hour until the
bromphenol blue dye front migrated to 3.0 cm off-origin. The top 1.5
cm of the gel was removed and the remaining gel was exposed at least
30 overnight with two intensification screens at -70C.
V. Discussion of Results
The autoradiograph showed that the AIDS DNA sequence was
only present in sample 368H, which was later found to be the only
12~924~i
2~
HTLVIII positive DNA. The other ten samples were as follows: (a)
194BK = DNA frr,m leukemia patient (no virus isolated), (b) 342 =
HTLVI, (c) 367 = HTLVI, (d) 361 = HTLVI, (e) 207 = patient with
aggressive leukemia (skin involvement), (f) 307 = HTLVI prototype cell
5 line (highest viral DNA to date), (9) 3088 = HTLVI, (h) 323 = HTLVII,
- (i) 326 = HTLVI, and (j) 340 = patient with aggressive leukemia(different from (e)).
Therefore, the primers employed were able to amplify the DNA
to allow the probe to detect accurately the sequence. The other
10 samples remained negative even with ten additional cycles of
amplification. Amplification in the presence of 10% DMS0 (minimizes
secondary structure formation) at 37C also indicated the HTLVIII
sample as the only positive sample.
EXAMPLE 2
In this example, the same procedure was followed as
described in Example 1 except that the primers employed, designated
SK24 and SK18, were as follows:
5'-ATCCCAGTAGGAGAA-3' (SK24)
5'-TTATGTCCAGAATGC-3' (SK18)
20 An alternative to SK24 was the primer SK25 as follows:
5'-ATAATCCACCTATCCCAG-3' (SK25)
The probe employed, SK19, was of the sequence:
5'-*ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC-3',
where * indicates the label. The probe was labeled as described in
25 Section III. SK24 and SK18 were selected to provide for the
amplification of a hydrophilic region of gag from nucleotides 1552 to
1642 of HTLVIII. SK25 and SK1~ were selected to provide for
amplifying a hydrophilic region of gag from nucleotides 1541 to 1642
of HTLVIII. SK19, when annealed to the amplified DNA, reconstitutes a
30 BstNI site. Digestion with this enzyme releases a 4-mer.
1~79'~4~
29
The autoradiograph for hybridization and restriction at 53C
showed as positive only the HTLVIII sample found in Example 1. A
background band appearing in all samples disappeared when the
temperature of hybridization and restriction was raised to 60C from
53C. The increased temperature is presumed to have minimized non-
_ specific hybridization of the probe.
EXAMPLE 3
A 180 bp DNA fragment which encodes a hydrophilic region of
gag (nucleotides 1470-1649 of HTLVIII-isolate B~10), having 186 base
pairs including BamHI ends, was constructed by first ligating 10
10 overlapping oligomers using T4 DNA ligase. The oligomers are shown in
Figure 2. The resulting fragment was then cloned into the BamHI site
of M13mplOw which is commercially available. None of the clones
sequenced had the exact desired sequence. However, using two clones
shown in Figure 3 (where X denotes a mutation in the gene), a clone
15 with the correct sequence was constructed by substituting the
SpeI/BstXI fragment of clone M13mplOW:C7 with the SpeI/BstXI fragment
of clone M13mplOW:D6. DNA from M13mplOW:C7 was digested with SpeI and
B XI, treated with alkaline phosphatase and the larger vector
containing fragment purified from an agarose gel. The SpeI/BstXI DNA
20 fragment from clone M13mplOW:D6 was purified from a polyacrylamide gel
after double digestions with the same enzymes. The purified fragments
from M13mplOW:C7 and M13mplOW:D6 were ligated and transformed into E.
coli strain DG98 available from the American Type Culture
Collection. The resulting clone, designated M-13-GAG, contains the
25 correct sequence and was deposited with the ATCC on January 8, 1986
with ATCC No. 40,218.
This M-13-GAG may be used to construct a gapped circle probe
as described above to evaluate an amplified sample in a non-isotopic
dot blot format.
The amplified product may be prepared from two primers, SK23
and SK28, which encompass the entire 180 mer in M-13-GAG and have the
following sequence:
1~79~44
5'-~TGAGAGAACCAAGG-3' (SK23)
5'-CCTT~TCTTATGTCCAG-3' (SK2~)
These primers, which were selected to amplify between nucleotides 1468
and 1649, were already tested with probe SK19 and found to detect the
HTLVIII samp1e successfully.
EXAMPLE 4
Seventy-one coded samples, the DNA of which was extracted by
Dr. Poiesz using the method described in Example 2 were initially
analyzed by their DNA using the primer pair of SK01 and SK02 of
Example 1 or SK17 and SK18, where SK18 is defined in Example 2 and
SK17 has the sequence:
5'-CCAGTAGGAGAAAT-3'
which is selected to amplify the hydrophilic region Of 9~1 from
nucleotides 1555 to 1642 of HTLVIII. The amplification using the
primer pair SK17 and SK18 was carried out in the presence of 10~ D~S0
by weight at 37C, but otherwise according to the procedure of Example
1. After amplification the procedure of Example 1 was used to detect
the DNA, using the probe SK03 (of Example 1) or SK19 (of Example 2).
Some of the ambiguous samples were further analyzed using the primer
pair SK24 and SK18 of Example 2 and using the probe SK19 at room
temperature.
The results show that all samples which were identified as
positive by the test herein were DNAs isolated from AIDS or ARC
patients, including a HTLVIII isolate, a LAV isolate and an AIDS-
25 associated virus (AAV) identified by Dr. Poiesz. In addition, anantibody positive, reverse transcriptase negative, healthy homosexual
who has had multiple contacts with AIDS victims was also identified as
positive by both sets of primer pairs. The SK17-SK18 and SK24-SK18
primer pairs appeared to detect more positives than the SK01-SK02
30 primer pair.
None of the negative control samples (normal T cells,
uninfected cell 1ines or HTLYII) showed positively in the assays
herein. The test herein identified ten infected samples as positive
~:7~4
31
which were negative by reverse transcriptase. On the other hand, five
samples which were reverse transcriptase positive (3 out of the 5 were
~) were negative by the test herein. All 71 samples proved to be
ELISA positive, showing that ELISA is not a very discriminative or
s specific test for the AIDS virus.
- The above examples show that AIDS viral DNA sequences can he
identified in cell lines infected with blood, semen mononuclear cells,
and semen supernatants from patients with AIDS or ARC.
EXAMPLE 5
This example illustrates that the technique herein can be
applied directly to the identification of AIDS viral DNA sequences in
peripheral mononuclear cells of fresh blood without having to
cult7vate the virus first.
Coded blood samples from AIDS patients were treated as
15 follows by Dr. Poeisz: First, they were centrifuged using low-speed
centrifugation (about 3000 x 9) to pellet all the cells, thereby
obtaining buffy coats. The buffy coats were passed through a Ficoll-
Hypaque density column and the leukocytes were collected from the
column. The DNA was extracted from the leukocytes by the procedure
20 described in Example 1.
The DNA was amplified using the primer pairs SK17 and SKl~
described in Examp1es 2 and 4, respectively, in the presence of 10%
DMSO by weight at 37C using the procedure described in Example 1 with
1 unit, 2 units and 4 units of Klenow fragment. After amplification,
25 the probes used, following the procedure of Example 1, were either
SK03 (of Example 1) or SKl9 (of Example 2). Some of the ambiguous
samples were further analyzed using the primer pair SK24 and SK18 of
Example 2 and the probe SKl9 at room temperature.
The results after overnight exposure show that the DNAs
30 isolated from some of the AIDS or ARC patients were identified as
positive.
127~4~
32
The experiment was repeated using primer pairs SK23 and SK28
(described in Example 3), and probe SK19 (Example 2). The experiment
was again repeated using primer pairs SK32 and SK33 having the
sequences as follows:
5'-ACCTGCCACCTGTAGTAG-3' (SK32)
S'-GCCATATTCCTGGACTACAG-3' (SK33)
and using the probe SK34 of the sequence:
5'-TAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGAGAAGCC-3' (SK34)
The restriction enzyme PvuII was used to cleave the restriction site
of SK34.
The results from both experiments after a 6-day exposure
period revealed that the DNAs iso1ated from some of the AIDS or ARC
patients were identified as positive.
EXAMPLE 6
The desired sequences to be amplified were contained in a
woodchuck hepatitis viral molecular clone from Chuck Rogler of Albert
Einstein College of Medicine and a human hepatitis B clone of the adw2
subtype with the insert pHBV1 in pBR322 obtained from Stanford
University and described by Sninsky et al., Nature, 279:346-348
(1979).
The sequences to be amplified, the primers, and the probes
were identified by the dot matrix program as described above, wherein
the sequence window selected was at least 20 base pairs long, so that
the sequences were chosen within conserved regions of the
hepadnaviruses. Regions of 20 contiguous bases of homology were
located after pairwise comparisons of the sequenced viral genomes and
variants thereof.
I. Synthesis of Primers
The following two oligodeoxyribonucleotide primers,
30 designated MD03 and MD06, respectively, were prepared by the method
described in Example 1, Section I:
~Z79~4~
5'-CTCAAGCTTCATCATCCATATA-3' (MD03)
5'-CTTGGATCCTATGGGAGTGG-3' (MD06)
These primers were selected from the polymerase gene of the
hepadnaviruses.
_ 5 II. Amplification Reaction
Ten pmoles of each plasmid was added to 100 ~l buffer
consisting of 10 mM Tris-HCl, pH 7.5, 50 mM sodium chloride and 10 mM
magnesium chloride and containing 100 picomoles of Primer MC03, 100
picomoles of Primer MD06, 10% by weight DMS0, and 150 nanomoles each
10 of dATP, dCTP, dGTP and TTP.
The treatment of this resulting solution occurred as
described in Example I, Section II, using 25 cycles, except at 37C
instead of room temperature.
III. Synthesis and Phosphorylation of Probe
A labeled DNA probe, MD09, of the sequence:
5'-*GGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTC-3'
where * indicates the label, was synthesized according to the
procedures described in Example 1, Section I. The probe was labeled
as described in Example 1, Section III.
20 IV. Hybridization/Digestion with Probe
The procedure of Example 1, Section IV was followed for
hybridization and digestion, except that DdeI was used as the
restriction enzyme to cleave the probe.
V, Results
The autoradiogram shows that signals are generated using the
primers herein as compared to a control (SC-1, which was deposited
with the ATCC on March 19, 19~5 and is an EBY-transformed B cell line
homozygous for the sick1e cell allele with no hepatitis genome), where
no amplification took place. Therefore, amplification of the
30 hepadnaviruses is possible using the technique herein.
127~44
3~
The results were the same when the above experiments were
performed using, instead of primers MD06 and MD03, the primers MD14
and MD13, as follows:
5'-GCGGG~TCCC~TCTTCTTATTGGTTCTTCTGG-3' (MD14) and
5'-GCGAAGCTTGTTAGGGTTTAAATGTATACCC-3' (MD13).
- These primers were selected from the envelope gene of the
hepadnaviruses.
The following deposit was made on the date given:
Strain Deposit Date ATCC No.
10 M13-GAG January 8, 1986 40,218
This deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure and the
2egulations thereunder (Budapest Treaty). This assures maintenance of
a viable culture for 30 years from date of deposit. The organism will
be made available by ATCC under the terms of the Budapest Treaty, and
subject to an agreement between applicants and ATCC, which assures
permanent and unrestricted availability of the progeny of the cultures
to the public upon issuance of the pertinent U.S. patent or upon
20 laying open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to one
determined by the U.S. Commissioner of Patents and Trademarks to be
entitled thereto according to 35 USC 122 and the Commissioner's rules
pursuant theretu (including 37 CF~ ~1.14 with particular reference to
25 ~6 OG 638). The assignee of the present application agrees that if
the culture on deposit should die or be lost or destroyed when
cultivated under suitable conditions, it will be promptly replaced on
notification with a viable specimen of the same culture. Availability
of the deposited strain is not to be construed as a license to
30 practice the invention in contravention of the rights granted under
the authority of any government in accordance with its patent laws.
