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Description  |
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This invention relates generally to the field of nucleic acid chemistry.
More specifically, it relates to the use of the 5' to 3' nuclease activity
of a nucleic acid polymerase to degrade a labeled oligonucleotide in a
hybridized duplex composed of the labeled oligonucleotide and a target
oligonucleotide sequence and form detectable labeled fragments.
Investigational microbiological techniques are routinely applied to
diagnostic assays. For example, U.S. Pat. No. 4,358,535 discloses a method
for detecting pathogens by spotting a sample (e.g., blood, cells, saliva,
etc.) on a filter (e.g., nitrocellulose), lysing the cells, and fixing the
DNA through chemical denaturation and heating. Then, labeled DNA probes
are added and allowed to hybridize with the fixed sample DNA,
hybridization indicating the presence of the pathogen's DNA. The sample
DNA in this case may be amplified by culturing the cells or organisms in
place on the filter.
A significant improvement in DNA amplification, the polymerase chain
reaction (PCR) technique, is disclosed in U.S. Pat. Nos. 4,683,202;
4,683,195; 4,800,159; and 4,965,188. In its simplest form, PCR is an in
vitro method for the enzymatic synthesis of specific DNA sequences, using
two oligonucleotide primers that hybridize to opposite strands and flank
the region of interest in the target DNA. A repetitive series of reaction
steps involving template denaturation, primer annealing, and the extension
of the annealed primers by DNA polymerase results in the exponential
accumulation of a specific fragment whose termini are defined by the 5'
ends of the primers. PCR is capable of producing a selective enrichment of
a specific DNA sequence by a factor of 109. The PCR method is also
described in Saiki et al., 1985, Science 230:1350.
Detection methods generally employed in standard PCR techniques use a
labeled probe with the amplified DNA in a hybridization assay. For
example, EP Publication No. 237,362 and PCT Publication No. 89/11548
disclose assay methods wherein the PCR-amplified DNA is first fixed to a
filter, and then a specific oligonucleotide probe is added and allowed to
hybridize. Preferably, the probe is labeled, e.g., with .sup.32 P, biotin,
horseradish peroxidase (HRP), etc., to allow for detection of
hybridization. The reverse is also suggested, that is, the probe is
instead bound to the membrane, and the PCR-amplified sample DNA is added.
Other means of detection include the use of fragment length polymorphism
(PCR-FLP), hybridization to allele-specific oligonucleotide (ASO) probes
(Saiki et al., 1986, Nature 324:163), or direct sequencing via the dideoxy
method using amplified DNA rather than cloned DNA. The standard PCR
technique operates essentially by replicating a DNA sequence positioned
between two primers, providing as the major product of the reaction a DNA
sequence of discrete length terminating with the primer at the 5' end of
each strand. Thus, insertions and deletions between the primers result in
product sequences of different lengths, which can be detected by sizing
the product in PCR-FLP. In an example of ASO hybridization, the amplified
DNA is fixed to a nylon filter (by, for example, UV irradiation) in a
series of "dot blots", then allowed to hybridize with an oligonucleotide
probe labeled with HRP under stringent conditions. After washing,
tetramethylbenzidine (TMB) and hydrogen peroxide are added: HRP catalyzes
the hydrogen peroxide oxidation of TMB to a soluble blue dye that can be
precipitated, indicating hybridized probe.
While the PCR technique as presently practiced is an extremely powerful
method for amplifying nucleic acid sequences, the detection of the
amplified material requires additional manipulation and subsequent
handling of the PCR products to determine whether the target DNA is
present. It would be desirable to decrease the number of subsequent
handling steps currently required for the detection of amplified material.
A "homogeneous" assay system, that is, one which generates signal while
the target sequence is amplified, requiring minimal post-amplification
handling, would be ideal.
The present invention provides a process for the detection of a target
nucleic acid sequence in a sample, said process comprising:
(a) contacting a sample comprising single-stranded nucleic acids with an
oligonucleotide containing a sequence complementary to a region of the
target nucleic acid and a labeled oligonucleotide containing a sequence
complementary to a second region of the same target nucleic acid strand,
but not including the nucleic acid sequence defined by the first
oligonucleotide, to create a mixture of duplexes during hybridization
conditions, wherein the duplexes comprise the target nucleic acid annealed
to the fast oligonucleotide and to the labeled oligonucleotide such that
the 3' end of the first oligonucleotide is adjacent to the 5' end of the
labeled oligonucleotide;
(b) maintaining the mixture of step (a) with a template-dependent nucleic
acid polymerase having a 5' to 3' nuclease activity under conditions
sufficient to permit the 5' to 3' nuclease activity of the polymerase to
cleave the annealed, labeled oligonucleotide and release labeled
fragments; and
(c) detecting and/or measuring the release of labeled fragments.
This process is especially suited for analysis of nucleic acid amplified by
PCR. This process is an improvement over known PCR detection methods
because it allows for both amplification of a target and the release of a
label for detection to be accomplished in a reaction system without resort
to multiple handling steps of the amplified product. Thus, in another
embodiment of the invention, a polymerase chain reaction amplification
method for concurrent amplification and detection of a target nucleic acid
sequence in a sample is provided. This method comprises:
(a) providing to a PCR assay containing said sample, at least one labeled
oligonucleotide containing a sequence complementary to a region of the
target nucleic acid, wherein said labeled oligonucleotide anneals within
the target nucleic acid sequence bounded by the oligonucleotide primers of
step (b);
(b) providing a set of oligonucleotide primers, wherein a first primer
contains a sequence complementary to a region in one strand of the target
nucleic acid sequence and primes the synthesis of a complementary DNA
strand, and a second primer contains a sequence complementary to a region
in a second strand of the target nucleic acid sequence and primes the
synthesis of a complementary DNA strand; and wherein each oligonucleotide
primer is selected to anneal to its complementary template upstream of any
labeled oligonucleotide annealed to the same nucleic acid strand;
(c ) amplifying the target nucleic acid sequence employing a nucleic acid
polymerase having 5' to 3' nuclease activity as a template-dependent
polymerizing agent under conditions which are permissive for PCR cycling
steps of (i) annealing of primers and labeled oligonucleotide to a
template nucleic acid sequence contained within the target region, and
(ii) extending the primer, wherein said nucleic acid polymerase
synthesizes a primer extension product while the 5' to 3' nuclease
activity of the nucleic acid polymerase simultaneously releases labeled
fragments from the annealed duplexes comprising labeled oligonucleotide
and its complementary template nucleic acid sequences, thereby creating
detectable labeled fragments; and
(d) detecting and/or measuring the release of labeled fragments to
determine the presence or absence of target sequence in the sample.
FIG. 1 is an autoradiograph of a DEAE cellulose thin layer chromatography
(TLC) plate illustrating the release of labeled fragments from cleaved
probe.
FIG. 2 is an autoradiograph of DEAE cellulose TLC plates illustrating the
thermostability of the labeled probe.
FIGS. 3A and 3B are autoradiographs of DEAE cellulose TLC plates showing
that the amount of labeled probe fragment released correlates with an
increase in PCR cycle number and starting template DNA concentration.
FIG. 4 illustrates the polymerization independent 5'-3' nuclease activity
of Taq DNA polymerase shown in the autoradiograph using a series of
primers which anneal from zero to 20 nucleotides upstream of the probe.
FIG. 5 is an autoradiograph showing the release of labeled probe fragments
under increasing incubation temperatures and time, wherein the composition
at the 5' end of the probe is GC rich.
FIG. 6 is an autoradiograph showing the release of labeled probe fragments
under increasing incubation temperatures and time, wherein the composition
at the 5' end of the probe is AT rich.
FIGS. 7A and 7B provide 5% acrylamide electrophoresis gel analysis of a 142
base pair HIV product, amplified in the presence or absence of labeled
probe.
FIGS. 8A and 8B are a composite of two autoradiographs of TLC analysis of
aliquots of PCR amplification products which show that radiolabel release
occurs and increases in amount with both increases in starting template
and with longer thermocycling.
FIG. 9 is a schematic for a reaction in which an NHS-active ester
derivative of biotin is added to the 3'-amine of an oligonucleotide probe.
FIG. 10 is a schematic for a reaction in which a biotin hydrazide is used
to label an oligonucleotide probe that has a 3'-ribonucleotide.
FIGS. 11A-11C show a schematic for labeling an oligonucleotide probe with
biotin using a biotin phosphoramidite.
FIG. 12 shows a reagents for labeling oligonucleotide probes with biotin.
FIG. 13 shows an oligonucleotide probe labeled with rhodamine-X-590 and
crystal violet.
FIG. 14 shows a schematic for a reaction to generate an active acyl azide
of crystal violet.
FIG. 15 shows a schematic for a reaction to add an amine to a thymidine for
use in conjugating a label to an oligonucleotide probe.
FIG. 16 shows typical results and relation of signal to input target number
for the present method using Bakerbond.TM. PEI solid phase extractant.
As used herein, a "sample" refers to any substance containing or presumed
to contain nucleic acid and includes a sample of tissue or fluid isolated
from an individual or individuals, including but not limited to, for
example, skin, plasma, serum, spinal fluid, lymph fluid, synovial fluid,
urine, tears, blood cells, organs, tumors, and also to samples of in vitro
cell culture constituents (including but not limited to conditioned medium
resulting from the growth of cells in cell culture medium, recombinant
cells and cell components).
As used herein, the terms "nucleic acid", "polynucleotide" and
"oligonucleotide" refer to primers, probes, oligomer fragments to be
detected, oligomer controls and unlabeled blocking oligomers and shall be
generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to
polyribonucleotides (containing D-ribose), and to any other type of
polynucleotide which is an N-glycoside of a purine or pyrimidine base, or
modified purine or pyrimidine bases. There is no intended distinction in
length between the term "nucleic acid", "polynucleotide" and
"oligonucleotide", and these terms will be used interchangeably. These
terms refer only to the primary structure of the molecule. Thus, these
terms include double- and single-stranded DNA, as well as double- and
single-stranded RNA. The oligonucleotide is comprised of a sequence of
approximately at least 6 nucleotides, preferably at least about 10-12
nucleotides, and more preferably at least about 15-20 nucleotides
corresponding to a region of the designated nucleotide sequence.
"Corresponding" means identical to or complementary to the designated
sequence.
The oligonucleotide is not necessarily physically derived from any existing
or natural sequence but may be generated in any manner, including chemical
synthesis, DNA replication, reverse transcription or a combination
thereof. The terms "oligonucleotide" or "nucleic acid" intend a
polynucleotide of genomic DNA or RNA, cDNA, semisynthetic, or synthetic
origin which, by virtue of its origin or manipulation: (1) is not
associated with all or a portion of the polynucleotide with which it is
associated in nature; and/or (2) is linked to a polynucleotide other than
that to which it is linked in nature; and (3) is not found in nature.
Because mononucleotides are reacted to make oligonucleotides in a manner
such that the 5' phosphate of one mononucleotide pentose ring is attached
to the 3' oxygen of its neighbor in one direction via a phosphodiester
linkage, an end of an oligonucleotide is referred to as the "5' end" if
its 5' phosphate is not linked to the 3' oxygen of a mononucleotide
pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5'
phosphate of a subsequent mononucleotide pentose ring. As used herein, a
nucleic acid sequence, even if internal to a larger oligonucleotide, also
may be said to have 5' and 3' ends.
When two different, non-overlapping oligonucleotides anneal to different
regions of the same linear complementary nucleic acid sequence, and the 3'
end of one oligonucleotide points toward the 5' end of the other, the
former may be called the "upstream" oligonucleotide and the latter the
"downstream" oligonucleotide.
The term "primer" may refer to more than one primer and refers to an
oligonucleotide, whether occurring naturally, as in a purified restriction
digest, or produced synthetically, which is capable of acting as a point
of initiation of synthesis along a complementary strand when placed under
conditions in which synthesis of a primer extension product which is
complementary to a nucleic acid strand is catalyzed. Such conditions
include the presence of four different deoxyribonucleoside triphosphates
and a polymerization-inducing agent such as DNA polymerase or reverse
transcriptase, in a suitable buffer ("buffer" includes substituents which
are cofactors, or which affect pH, ionic strength, etc.), and at a
suitable temperature. The primer is preferably single-stranded for maximum
efficiency in amplification.
The complement of a nucleic acid sequence as used herein refers to an
oligonucleotide which, when aligned with the nucleic acid sequence such
that the 5' end of one sequence is paired with the 3' end of the other, is
in "antiparallel association." Certain bases not commonly found in natural
nucleic acids may be included in the nucleic acids of the present
invention and include, for example, inosine and 7-deazaguanine.
Complementarity need not be perfect; stable duplexes may contain
mismatched base pairs or unmatched bases. Those skilled in the an of
nucleic acid technology can determine duplex stability empirically
considering a number of variables including, for example, the length of
the oligonucleotide, base composition and sequence of the oligonucleotide,
ionic strength, and incidence of mismatched base pairs.
Stability of a nucleic acid duplex is measured by the melting temperature,
or "T.sub.m "The T.sub.m of a particular nucleic acid duplex under
specified conditions is the temperature at which half of the base pairs
have disassociated.
As used herein, the term "target sequence" or "target nucleic acid
sequence" refers to a region of the oligonucleotide which is to be either
amplified, detected or both. The target sequence resides between the two
primer sequences used for amplification.
As used herein, the term "probe" refers to a labeled oligonucleotide which
forms a duplex structure with a sequence in the target nucleic acid, due
to complementarity of at least one sequence in the probe with a sequence
in the target region. The probe, preferably, does not contain a sequence
complementary to sequence(s) used to prime the polymerase chain reaction.
Generally the 3' terminus of the probe will be "blocked" to prohibit
incorporation of the probe into a primer extension product. "Blocking" can
be achieved by using non-complementary bases or by adding a chemical
moiety such as biotin or a phosphate group to the 3' hydroxyl of the last
nucleotide, which may, depending upon the selected moiety, serve a dual
purpose by also acting as a label for subsequent detection or capture of
the nucleic acid attached to the label. Blocking can also be achieved by
removing the 3'-OH or by using a nucleotide that lacks a 3'-OH such as a
dideoxynucleotide.
The term "label" as used herein refers to any atom or molecule which can be
used to provide a detectable (preferably quantifiable) signal, and which
can be attached to a nucleic acid or protein. Labels may provide signals
detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray
diffraction or absorption, magnetism, enzymatic activity, and the like.
As defined herein, "5'.fwdarw.3' nuclease activity" or "5' to 3' nuclease
activity" refers to that activity of a template-specific nucleic acid
polymerase including either a 5'.fwdarw.3' exonuclease activity
traditionally associated with some DNA polymerases whereby nucleotides are
removed from the 5' end of an oligonucleotide in a sequential manner,
(i.e., E. coli DNA polymerase I has this activity whereas the Klenow
fragment does not), or a 5'.fwdarw.3' endonuclease activity wherein
cleavage occurs more than one phosphodiester bond (nucleotide) from the 5'
end, or both.
The term "adjacent" as used herein refers to the positioning of the primer
with respect to the probe on its complementary strand of the template
nucleic acid. The primer and probe may be separated by 1 to about 20
nucleotides, more preferably, about 1 to 10 nucleotides, or may directly
abut one another, as may be desirable for detection with a
polymerization-independent process. Alternatively, for use in the
polymerization-dependent process, as when the present method is used in
the PCR amplification and detection methods as taught herein, the
"adjacency" may be anywhere within the sequence to be amplified, anywhere
downstream of a primer such that primer extension will position the
polymerase so that cleavage of the probe occurs.
As used herein, the term "thermostable nucleic acid polymerase" refers to
an enzyme which is relatively stable to heat when compared, for example,
to nucleotide polymerases from E. coli and which catalyzes the
polymerization of nucleoside triphosphates. Generally, the enzyme will
initiate synthesis at the 3'-end of the primer annealed to the target
sequence, and will proceed in the 5'-direction along the template, and if
possessing a 5' to 3' nuclease activity, hydrolyzing intervening, annealed
probe to release both labeled and unlabeled probe fragments, until
synthesis terminates. A representative thermostable enzyme isolated from
Thermus aquaticus (Taq) is described in U.S. Pat. No. 4,889,818 and a
method for using it in conventional PCR is described in Saiki et al.,
1988, Science 239:487.
Taq DNA polymerase has a DNA synthesis-dependent, strand replacement 5'-3'
exonuclease activity (see Gelfand, "Tag DNA Polymerase" in PCR Technology;
Principles and Applications for DNA Amplification, Erlich, Ed., Stockton
Press, N.Y. (1989), Chapter 2). In solution, there is little, if any,
degradation of labeled oligonucleotides.
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of molecular biology, microbiology and
recombinant DNA techniques, which are within the skill of the art. Such
techniques are explained fully in the literature. See, e.g., Sambrook,
Fritsch & Maniatis, Molecular Cloning; A Laboratory Manual, Second Edition
(1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins, eds., 1984); A Practical Guide
to Molecular Cloning (B. Perbal, 1984); and a series, Methods in
Enzymology (Academic Press, Inc.). All patents, patent applications, and
publications mentioned herein, both supra and infra, are hereby
incorporated by reference.
The various aspects of the invention are based on a special property of
nucleic acid polymerases. Nucleic acid polymerases can possess several
activities, among them, a 5' to 3' nuclease activity whereby the nucleic
acid polymerase can cleave mononucleotides or small oligonucleotides from
an oligonucleotide annealed to its larger, complementary polynucleotide.
In order for cleavage to occur efficiently, an upstream oligonucleotide
must also be annealed to the same larger polynucleotide.
The 3' end of this upstream oligonucleotide provides the initial binding
site for the nucleic acid polymerase. As soon as the bound polymerase
encounters the 5' end of the downstream oligonucleotide, the polymerase
can cleave mononucleotides or small oligonucleotides therefrom.
The two oligonucleotides can be designed such that they anneal in close
proximity on the complementary target nucleic acid such that binding of
the nucleic acid polymerase to the 3' end of the upstream oligonucleotide
automatically puts it in contact with the 5' end of the downstream
oligonucleotide. This process, because polymerization is not required to
bring the nucleic acid polymerase into position to accomplish the
cleavage, is called "polymerization-independent cleavage."
Alternatively, if the two oligonucleotides anneal to more distantly spaced
regions of the template nucleic acid target, polymerization must occur
before the nucleic acid polymerase encounters the 5' end of the downstream
oligonucleotide. As the polymerization continues, the polymerase
progressively cleaves mononucleotides or small oligonucleotides from the
5' end of the downstream oligonucleotide. This cleaving continues until
the remainder of the downstream oligonucleotide has been destabilized to
the extent that it dissociates from the template molecule. This process is
called "polymerization-dependent cleavage."
In the present invention, a label is attached to the downstream
oligonucleotide. Thus, the cleaved mononucleotides or small
oligonucleotides which are cleaved by the 5'-3' nuclease activity of the
polymerase can be detected.
Subsequently, any of several strategies may be employed to distinguish the
uncleaved labeled oligonucleotide from the cleaved fragments thereof. In
this manner, the present invention permits identification of those nucleic
acid samples which contain sequences complementary to the upstream and
downstream oligonucleotides.
The present invention exploits this 5' to 3' nuclease activity of the
polymerase when used in conjunction with PCR. This differs from previously
described PCR amplification wherein the post-PCR amplified target
oligonucleotides are detected, for example, by hybridization with a probe
which forms a stable duplex with that of the target sequence under
stringent to moderately stringent hybridization and wash conditions. In
contrast to those known detection methods used in post-PCR amplifications,
the present invention permits the detection of the target nucleic acid
sequences during amplification of this target nucleic acid. In the present
invention, a labeled oligonucleotide is added concomitantly with the
primer at the start of PCR, and the signal generated from hydrolysis of
the labeled nucleotide(s) of the probe provides a means for detection of
the target sequence during its amplification.
The present invention is compatible, however, with other amplification
systems, such as the transcription amplification system, in which one of
the PCR primers encodes a promoter that is used to make RNA copies of the
target sequence. In similar fashion, the present invention can be used in
a self-sustained sequence replication (3SR) system, in which a variety of
enzymes are used to make RNA transcripts that are then used to make DNA
copies, all at a single temperature. By incorporating a polymerase with
5'.fwdarw.3' exonuclease activity into a ligase chain reaction (LCR)
system, together with appropriate oligonucleotides, one can also employ
the present invention to detect LCR products.
Of course, the present invention can be applied to systems that do not
involve amplification. In fact, the present invention does not even
require that polymerization occur. One advantage of the
polymerization-independent process lies in the elimination of the need for
amplification of the target sequence. In the absence of primer extension,
the target nucleic acid is substantially single-stranded. Provided the
primer and labeled oligonucleotide are adjacently bound to the target
nucleic acid, sequential rounds of oligonucleotide annealing and cleavage
of labeled fragments can occur. Thus, a sufficient amount of labeled
fragments can be generated, making detection possible in the absence of
polymerization. As would be appreciated by those skilled in the art, the
signal generated during PCR amplification could be augmented by this
polymerization-independent activity.
In either process described herein, a sample is provided which is suspected
of containing the particular oligonucleotide sequence of interest, the
"target nucleic acid". The target nucleic acid contained in the sample may
be first reverse transcribed into cDNA, if necessary, and then denatured,
using any suitable denaturing method, including physical, chemical, or
enzymatic means, which are known to those of skill in the art. A preferred
physical means for strand separation involves heating the nucleic acid
until it is completely (>99%) denatured. Typical heat denaturation
involves temperatures ranging from about 80.degree. C. to about
105.degree. C., for times ranging from a few seconds to minutes. As an
alternative to denaturation, the target nucleic acid may exist in a
single-stranded form in the sample, such as, for example, single-stranded
RNA or DNA viruses.
The denatured nucleic acid strands are then incubated with preselected
oligonucleotide primers and labeled oligonucleotide (also referred to
herein as "probe") under hybridization conditions, conditions which enable
the binding of the primers and probes to the single nucleic acid strands.
As known in the art, the primers are selected so that their relative
positions along a duplex sequence are such that an extension product
synthesized from one primer, when the extension product is separated from
its template (complement), serves as a template for the extension of the
other primer to yield a replicate chain of defined length.
Because the complementary strands are longer than either the probe or
primer, the strands have more points of contact and thus a greater chance
of finding each other over any given period of time. A high molar excess
of probe, plus the primer, helps tip the balance toward primer and probe
annealing rather than template rean | | |
