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Claims  |
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We claim:
1. In a method for a polymerase dependent extension reaction for
synthesizing an extension product complementary to a target nucleic acid
within a larger nucleic acid template comprising:
combining a pair of primers with said nucleic acid template;
and performing said polymerase dependent extension reaction under
conditions sufficient to synthesize said extension product, the
improvement comprising:
combining said pair of primers with the nucleic acid template in the
presence of at least one energy sink oligonucleotide that is incapable of
acting as a primer in an extension reaction, wherein each of said at least
one energy sink oligonucleotides has sufficient complementarity to at
least one of said primers and is present in sufficient concentration so as
to competitively inhibit binding of said primer to a non-target nucleic
acid via hybridization of said energy sink oligonucleotide to said primer.
2. The method of claim 1, wherein the 3' end of at least one of said
primers protrudes when hybridized to said energy sink oligonucleotide.
3. The method of claim 1, wherein the energy sink oligonucleotide has at
least 10 consecutive bases perfectly complementary to a 10 base sequence
in the one of the primers.
4. The method of claim 1, wherein a lagging probe having a reporting group
attached thereto is hybridized to the nucleic acid template at a position
downstream of the direction of elongation, the method further comprising:
utilizing elongation of the primer to displace the lagging probe from the
template and
measuring a change in a signal resulting from the displacement of the
lagging probe from the nucleic acid template.
5. The method of claim 1, wherein a lagging probe is hybridized to the
nucleic acid template at a position downstream of the direction of
elongation, and wherein the primer and the lagging probe are labeled such
that the generation of a signal depends upon the juxtaposition of the
labelled primer and the labelled lagging probe, the method further
comprising:
utilizing elongation of the primer to displace the lagging probe from the
template and
measuring a change in a signal resulting from the displacement of the
lagging probe from the nucleic acid template.
6. A method for amplifying and detecting a target nucleic acid sequence in
a nucleic acid template in the same vessel, the method comprising:
a) providing in the presence of the nucleic acid template, a first duplex
and a second duplex, the first duplex including a first primer and a first
energy sink oligonucleotide, the second duplex including a second primer
and a second energy sink oligonucleotide, wherein the first energy sink
oligonucleotide is of sufficient complementarity to the first primer so as
to competitively inhibit binding of the first primer to a non-target
sequence in the nucleic acid template and wherein the second energy sink
oligonucleotide is of sufficient complementarity to the second primer so
as to competitively inhibit binding of the second primer to the non-target
sequence, wherein the first energy sink oligonucleotide and the second
energy sink oligonucleotide are incapable of acting as extension primers
in an extension reaction;
b) allowing the first primer, the first energy sink oligonucleotide, the
second primer and the second energy sink oligonucleotide to hybridize to
the template so that the first energy sink oligonucleotide hybridizes to
the template at a position downstream of the direction of elongation of
the second primer and the second energy sink oligonucleotide hybridizes to
the template at a position downstream of the direction of elongation of
the first primer, wherein at least one of the first energy sink
oligonucleotide and the second energy sink oligonucleotide has a reporting
group attached thereto;
c) subjecting the template having the primers and the energy sink
oligonucleotides hybridized thereto to conditions sufficient to permit the
first primer to elongate sufficiently to displace the second energy sink
oligonucleotide and the second primer to elongate sufficiently to displace
the first energy sink oligonucleotide from the template, and
d) measuring a change in a signal resulting from the displacement of the
energy sink oligonucleotide having the reporting group attached thereto
from the template.
7. The method of claim 6, wherein the reporting group is selected from the
group consisting of a fluorophore, a chromophore and a specific binding
agent.
8. The method of claim 7, wherein the reporting group is a fluorophore.
9. The method of claim 6, wherein at least one of the primers has a
reporting group attached thereto, wherein the primer having the reporting
group attached thereto and the energy sink oligonucleotide having the
reporting group attached thereto are labelled such that a first signal is
measurable when the labeled primer and the labeled energy sink
oligonucleotide are juxtaposed and a second signal is measurable when the
labeled primer and the labeled energy sink oligonucleotide are not
juxtaposed with one another.
10. The method of claim 9, wherein the reporting group attached to the
primer and the reporting group attached to the energy sink oligonucleotide
are fluorophores with overlapping emission and excitation wavelengths.
11. The method of claim 6, wherein at least one of the primers has a
reporting group attached thereto, wherein the primer having the reporting
group attached thereto and the energy sink oligonucleotide having the
reporting group attached thereto are labelled such that juxtaposition of
the labeled primer and the labeled energy sink oligonucleotide results in
quenching of a signal emitted by the reporting group attached to the
primer or by the reporting group attached to the energy sink
oligonucleotide.
12. A kit for use in a polymerase dependent extension reaction that
utilizes a pair of primers to synthesize an extension product
complementary to a target sequence in a nucleic acid template wherein
binding of at least one of the primers to a non-target sequence is
prevented, the kit consisting essentially of:
a duplex containing a primer and an energy sink oligonucleotide that is
incapable of acting as an extension primer in an extension reaction,
wherein the energy sink oligonucleotide is sufficiently complementary to
the primer to competitively inhibit binding of the primer to the
non-target sequence.
13. The kit of claim 12, wherein the energy sink oligonucleotide has a 5'
end, and wherein the 3' end of the extension primer protrudes beyond the
5' end of the energy sink oligonucleotide when the primer and the energy
sink oligonucleotide are hybridized to one another.
14. A kit for use in a polymerase dependent extension reaction that
utilizes a pair of primers to synthesize an extension product
complementary to a target sequence in a nucleic acid template wherein
binding of at least one of the primers to a non-target sequence is
prevented, the kit consisting essentially of:
a first duplex containing a first primer and a first energy sink
oligonucleotide that is incapable of acting as an extension primer in an
extension reaction, wherein the first energy sink oligonucleotide is
complementary to the first primer; and
a second duplex containing a second primer and a second energy sink
oligonucleotide that is incapable of acting as an extension primer in an
extension reaction, wherein the second energy sink oligonucleotide is
complementary to the second primer;
wherein the first energy sink oligonucleotide and the second energy sink
oligonucleotide competitively inhibit binding of the primers to the
non-target sequence, and further, wherein the first primer and the second
energy sink oligonucleotide each are labeled with a different reporting
group such that a first signal is measurable when the first primer and
second energy sink oligonucleotide are juxtaposed and wherein a second
signal is measurable when the first primer and the second energy sink
oligonucleotide are not juxtaposed with one another.
15. The method of claim 14, wherein juxtaposition of the labeled primer and
the labeled energy sink oligonucleotide results in quenching of a signal
emitted by the reporting group attached to the first primer or by the
reporting group attached to the second energy sink oligonucleotide.
16. The kit of claim 14, wherein both of the energy sink oligonucleotides
have a 5' end and wherein the 3' end of at least one of the primers
protrudes beyond the 5' end of its complementary energy sink
oligonucleotide when the primer and the energy sink oligonucleotide are
hybridized to one another. |
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Claims |
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Description |
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FIELD OF THE INVENTION
This invention relates to a process for nucleic acid amplification in the
absence of non-specific priming events. The invention also provides for
detection of amplification by utilizing extension product elongation to
displace a downstream probe. The invention further provides a homogeneous
process for simultaneously amplifying and detecting a target sequence in a
nucleic acid template. Products and apparatus related to the above-recited
processes are also provided.
BACKGROUND
Recent development of the polymerase chain reaction (PCR) has provided an
important tool for the detection of nucleic acid sequences present at low
concentrations (Mullis, K. B. et al., U.S. Pat. No. 4,683,195 and
4,683,202). In PCR, a segment of target sequence having boundaries defined
by two oligonucleotide extension primers, is amplified through repeated
enzymatic cycles to provide additional templates for further amplification
reactions. Accordingly, a small number of target sequences can be
exponentially amplified and readily detected. A major limitation of PCR
lies in the extensive generation of by-products produced as a result of
non-specific priming events, e.g., random priming of the nucleic acid
template and/or self priming of the extension primers. Thus, when a high
number of amplification cycles are required to amplify a target sequence
present at a relatively low concentration, the products of non-specific
priming events significantly impede PCR sensitivity.
An additional, related limitation of PCR is the requirement for a
separation step prior to detection of the amplified target. According to
standard PCR conditions, separation of the amplified target sequence from
the products of non-specific priming events is a prerequisite for
detection of the amplified target sequence. The absence of a homogenous
amplification reaction, i.e., a reaction in which amplification and
detection take place in the same reaction vessel has been an obstacle in
automating the PCR procedure. In addition, the requirement for a
separation step also subjects the PCR mixture to potential contamination
resulting from the separation procedure. The likelihood of contamination
severely limits the potential application of PCR in routine clinical
diagnosis.
Attempts have been reported to develop a homogeneous assay for
amplification and detection. One such attempt is described in the
procedure known as the ligase chain reaction (LCR, Beckman, K. C. and
Wang, C. N., European Pat. No. 320,308). LCR is performed using two pairs
of immediately adjacent and ligatable probes. The probes are amplified
through repeated cycles of ligation. However, the probes can randomly
ligate to each other to produce a background signal which is difficult to
eliminate, thus reducing the sensitivity of detection.
SUMMARY OF THE INVENTION
The deficiencies in the prior art are overcome by the present invention
which provides target amplification with substantially reduced
amplification of non-target extension product. As a result, a truly
homogenous process is achieved for simultaneously amplifying and detecting
a target sequence in a nucleic acid template without the requirement of a
separation step.
According to one aspect of the invention, a method for synthesizing
extension products corresponding to a target sequence within a larger
nucleic acid template is improved. A pair of primers for initiating
polymerization dependent extensions to form the extension products are
applied to the target sequence under conditions of polymerization
dependent extension. These conditions substantially reduce or even prevent
the synthesis of detectable amounts of non-target extension products. In
the preferred embodiments, the extension conditions include the
application of the primers to the template in the presence of an energy
sink of sufficient binding affinity to at least one of the primers and in
sufficient concentration so as to competitively inhibit binding of one of
the primers to nontarget sites. The energy sink may be an oligonucleotide
at least in part complementary to one of the primers and most preferably,
the energy sink is a pair of oligonucleotides, one each complementary to
the pair of extension primers. Detection of the extension product may be
achieved according to various methods in which one or both of the primers
and oligonucleotide are labelled.
According to another aspect of the invention, a homogeneous process for
amplifying a target sequence in a nucleic acid and detecting the presence
of the amplification without a separation step is provided. Two
complementary strands of target sequence are treated with a pair of
target-defining oligonucleotide primers, at least one of the primers being
labelled, and with a labelled oligonucleotide reporter molecule, the
reporter and primer producing a first signal when juxtaposed and a second
signal when remote from one another. Extension conditions then are applied
in repeated cycles to permit exponential amplification of the target
sequence, after which at least one of the signals is measured. The
reporter molecule may be adapted to prevent initiation from the reporter
molecule of non-target extension. The extension conditions are adapted to
reduce the likelihood of labelled reporter molecule from being juxtaposed
with primer after repeated cycles of extension, and the conditions also
may be such as to result in substantially-intact displacement of the
reporter molecule. Alternatively, the conditions may be such as to result
in some cleavage of labelled reporter molecule concurrent with the
extension reaction, so long as the cleaved fragment contains the label and
is displaced from its original position. The reporter molecule may be
either an energy sink probe or a lagging probe.
According to still another aspect of the invention, a method for detecting
a target sequence in a nucleic acid template is provided in which the
template is treated with an extension primer and a lagging probe.
Conditions then are applied to result in the formation of an extension
product having a sequence corresponding to the target sequence. Initiation
of extension product formation is by the extension primer. Elongation of
the extension product is utilized to displace the lagging probe in
substantially-intact condition. A signal generated by a reporting group
attached to at least one of the lagging probe and extension primer results
following displacement of the lagging probe and is measured.
In many of the methods of the invention, the extension primer may include
an allele-specific recognition sequence which typically is positioned
within two nucleotides of the 3' end of the extension primer.
According to still another aspect of the invention, a homogeneous process
based upon strand-displacement amplification, is provided for detecting at
least one target nucleic acid sequence. Two pairs of mutually
complementary nucleic acid probes are provided in the presence of target
nucleic acid. The probe pairs comprise a leading probe and a lagging
probe, each being sufficiently complementary to the target nucleic acid
sequence to hybridize therewith, the pairs of probes being constructed and
arranged such that when hybridized to the target nucleic acid, the leading
probes are juxtaposed relative to the 5' end of the lagging probes and
separated therefrom by at least one nucleotide to form a gap between the
leading and lagging probes of each probe pair. The probe pairs are then
hybridized with the nucleic acid and subjected to conditions sufficient to
permit the leading probe of each pair to elongate sufficiently to displace
the lagging probe of each pair, thereby forming a replica of the target
nucleic acid. Then, a reporting signal generated by the removal of lagging
probes is detected. Preferably, the conditions applied result in the
substantially-intact displacement of lagging probes. The reporting signal
may be generated by a molecule attached to one of the leading and lagging
probes, or both, the molecule being selected from the group consisting of
a fluorophore, a chromophore, and a specific binding agent. Most
preferably, the reporting signal is based upon the disruption of the
interaction between a leading and a lagging probe. For example, one of the
leading and one of the lagging probes may have fluorophores with
overlapping emission and excitation wavelengths, and the reporting signal
may be detected by measuring the disruption of the interaction between the
overlapping emission and excitation wavelengths.
Alternatively, specific binding agents may be used to generate a signal
dependent upon the relative proximity of probes. Specific binding agents
include molecular entities such as enzymes, e.g., .beta.-galactosidase,
ribonuclease S and alkaline phosphatase. Each of these enzymes is capable
of reassociating to form a functionally active enzyme following specific
enzyme cleavage. This ability to reassociate and restore functional
activity is referred to as intramolecular .alpha. complementation. For
example, a specific binding enzyme agent may be cleaved into a first
fragment and a second fragment. When the two fragments are proximately
located to one another, the enzyme reassociates and is capable of
catalyzing an enzyme reaction. For example, the first fragment may be
covalently attached to the 3' end of an extension primer and the second
fragment may be attached to the 5' end of an energy sink oligonucleotide
which is complementary to the extension primer. When the extension primer
is hybridized to the energy sink, the first and second fragments
reassociate to form a functionally active enzyme. Upon introduction of an
appropriate substrate and assay conditions, the enzyme will convert the
substrate to a product capable of detection, e.g., a colorimetric
substrate.
According to yet another aspect of the invention, a kit for use in the
amplification of a target sequence in a nucleic acid template is provided.
The kit includes a first oligonucleotide duplex including an extension
primer for initiating the synthesis of an extension product corresponding
to the target sequence in the nucleic acid template and a lagging probe
complementary to the extension primer, wherein the lagging probe is
adapted to prevent extension of non-target sequences. The extension primer
may have a 3' end protruding beyond the 5' end of the lagging probe. The
extension primer also may include an allele-specific recognition sequence
at one of its ends. The extension primer and the lagging probe may be
labelled in a complementing manner such that when they are juxtaposed,
they produce a first signal that differs from a second signal produced
when they are remote from one another. Most preferably, the kit includes a
second oligonucleotide duplex including a second extension primer and a
second lagging probe, wherein the first and second primers are
target-defining primers.
Still another aspect of the invention provides an apparatus for
simultaneously performing amplification of a target sequence in a nucleic
acid template and detecting the presence of the amplified target in the
absence of a separation step. The apparatus includes a probe container for
containing an oligonucleotide duplex and a reaction vessel for containing
a nucleic acid sample, the vessel is operatively linked to the probe
container for receiving the duplex contained therein. The apparatus
further has means for controlling mixing of the probes and sample and
means for controlling the temperature of the reaction vessel. The
apparatus also has a source for irradiating the reaction vessel and a
means for detecting radiation emitted from the reaction vessel. Most
preferably, the apparatus has a control mechanism which terminates the
amplification reaction when a pre-designated level of radiation is
detected by the means for detecting.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of the major steps constituting a
cycle of polymerization-dependent strand-displacement amplification.
FIG. 2 shows an autoradiogram of a polyacrylamide gel electrophoresis
(PAGE) gel demonstrating polymerization dependent strand-displacement as a
predominant phenomenon.
FIG. 3 shows an autoradiogram of native PAGE gel illustrating
polymerization dependent strand displacement during amplification.
FIG. 4A and FIG. 4B show an autoradiogram of a Urea-PAGE gel illustrating
distinct specificity for strand-displacement dependent amplification in
comparison to PCR method, using different polymerization agents.
FIG. 5A and 5B show the results of the homogeneous assay of the invention
for the detection of the HBV sequence. FIG. 5A is a line graph
illustrating the analysis of detecting different amounts of target HBV
sequence at various amplification cycle. FIG. 5B is a bar graph
illustrating the analysis of detecting different amounts of target HBV
sequence at cycle 40.
FIG. 6 shows an autoradiogram of PAGE gel illustrating the agreement
between the visual observation of product by strand-displacement
amplification and signal analysis of similar detection process presented
in FIG. 5A and FIG. 5B.
FIG. 7 shows the extension primer, lagging probe and template used in
connection with Example 1: Polymerization Dependent Strand-Displacement;
FIG. 8 shows pairs of extension primers and energy sink oligonucleotides
used in connection with Example 3: Energy Transfer for Fluorophore
Conjugated Probe Pairs;
FIG. 9 shows a four probe configuration used in connection with Example 5:
A Homogeneous Assay for Detection of HBV Sequence; and
FIG. 10 shows a three probe configuration used in connection with Example
5: A Homogeneous Assay for Detection of HBV Sequence.
FIG. 11 illustrates the configuration of a microtiter plate utilized in the
present invention;
FIG. 12 is a block diagram of one embodiment of the detector apparatus of
the present invention;
FIG. 13 illustrates the physical construction of a bifurcated optical cable
utilized by the present invention;
FIG. 14 is a flowchart illustrating a method by which the detection
apparatus of the present invention determines the fluorescence level
within each well in the microtiter plate;
FIG. 15 is a block diagram of one embodiment of the amplification/detection
apparatus of the present invention;
FIG. 16 is a block diagram of a temperature control unit utilized by the
amplification/detection apparatus of the present invention;
FIG. 17 is a flowchart illustrating a method of determining an initial
fluorescence ratio for each well within a microtiter plate;
FIG. 18 is a flowchart illustrating subroutine Readwells which is a method
for reading the fluorescent level of every well in the microtiter plate;
FIG. 19 is a flowchart illustrating the beginning steps of the detection
method of the present invention;
FIG. 20 is a flowchart that is a continuation of the flowchart illustrated
in FIG. 19 and illustrates the remaining steps of the detection method of
the present invention;
FIG. 21 is a flowchart illustrating the beginning steps of the
amplification/detection method of the present invention;
FIG. 22 is a flowchart that is a continuation of FIG. 21 and illustrates
further steps of the amplification/detection method of the present
invention;
FIG. 23 is a flowchart that is a continuation of the flowchart in FIG. 22
and illustrates additional steps of the amplification/detection method of
the present invention; and
FIG. 24 is a flowchart that is a continuation of the flowchart in FIG. 23
and illustrates the remaining steps of the amplification/detection method
of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
In a preferred embodiment, an improved method is provided for synthesizing
complementary pairs of extension products corresponding to a target
sequence in a nucleic acid template. The improvement comprises utilizing
an energy sink oligonucleotide to inhibit non-specific priming events.
As used herein, nucleic acid amplification refers broadly to a process for
producing any particular nucleic acid sequence, i.e., the "target
sequence", in amounts which are large compared to the amount initially
present. One such process is described in U.S. Pat. No. 4,683,202, the
contents of which are incorporated herein by reference. However, U.S. Pat.
No. 4,683,202 does not address elimination of non-specific priming events
occurring during nucleic acid amplification reactions; nor does the
recited patent describe a homogeneous assay for simultaneously amplifying
a target sequence and detecting the extension products corresponding
thereto.
As in other amplification schemes, extension primers define the boundaries
of the specific target sequence. The target 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 HBV gene contained in human genomic DNA
(Example 5) or a portion of a nucleic acid sequence of a particular
microorganism. The organism may constitute only a minor fraction of a
particular biological sample.
Target defining oligonucleotide primers means primers, each complementary
with a different strand of two separate complementary nucleic acid
strands, such that when an extension product of one primer in the
direction of the other primer is generated, that extension product can
serve as a template for the synthesis of the extension product of the
other primer. The target sequence is that portion of the nucleic acid
between and including the sequence corresponding to the primers.
The phrase "non-specific priming events" refers to events which lead to
amplification of a sequence on the template other than the target
sequence. Non-specific priming events include reactions such as the random
hybridization of an extension primer to a "non-priming sequence" of the
template and self priming (inter and intra molecular reactions) by the
extension primers. By "non-priming sequence" is meant a sequence on the
template to which the extension primer may non-specifically hybridize,
thereby initiating amplification of a non-target sequence. Non-specific
priming events are a fairly common occurrence during amplification, due to
the ability of the extension primer to hybridize to itself, other
extension primers or to sequences on the template to which the primer is
only partially complementary. Thus, as used herein, the term "non-target
sequence" refers broadly to those sequences which are not target
sequences, i.e., those sequences which are not the desired target or
object of the amplification reaction. Such non-target sequences are
unintentionally amplified by the non-specific hybridization of an
extension primer to a non-priming sequence.
The nucleic acid template may contain more than one target sequence. Like
the invention of U.S. Pat. No. 4,683,202, the present invention is useful
for producing large amounts of one target sequence as well as for
simultaneously amplifying more than one different target sequence located
on the same or different nucleic acid templates. However, the present
invention advantageously reduces to nondetectable amounts, non-specific
priming events, thus making possible the simultaneous amplification and
detection of a plurality of target sequences initially present in small
amounts in the template.
Amplification relies upon hybridization of an "extension primer" to a
specific "priming sequence" on the template. The terms "primer" or
"extension primer" refer 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 when
placed under conditions in which synthesis of a primer extension product
complementary to a target sequence is induced. Thus the phrase, "capable
of acting as a primer of an extension reaction" refers to the ability of
an oligonucleotide to act as a point of initiation for extension product
synthesis. Representative conditions for extension product synthesis are
provided in the Examples.
As used herein, the term "oligonucleotide" refers to a molecule consisting
of two or more deoxyribonucleotides or ribonucleotides, and preferably
containing more than three nucleotides. The size of the oligonucleotide
will depend on many factors, including the ultimate function or use of the
oligonucleotide. Preferably, an oligonucleotide which functions as an
extension primer will be sufficiently long to prime the synthesis of
extension products in the presence of a catalyst, e.g., DNA polymerase,
and deoxynucleotide triphosphates. The exact lengths of the primers will
depend on many factors, including temperature, source of primer and use of
the method. In diagnostic applications, for example, the oligonucleotide
primer typically contains 15-25 or more nucleotides, depending on the
complexity of the target sequence. For non-extension product applications,
the oligonucleotide generally contains between 10-25 nucleotides. Shorter
oligonucleotide generally require cooler temperatures to form sufficiently
stable hybrid complexes with template.
To initiate "specific amplification", the extension primer hybridizes to a
"priming sequence" on the template. Thus, the extension primer is designed
to have a sequence which is "substantially complementary" to that of the
priming sequence on the template. By "substantially complementary" between
the primer and the priming sequence is meant that the two sequences must
have a degree of nucleotide complementarity sufficient for the primer to
hybridize to the priming sequence and act as a point of initiation for
synthesis of an extension product.
Accordingly, the extension primer sequence is not required to be perfectly
complementary to the priming sequence of the template. For example, a
non-complementary nucleotide fragment may be attached to the 5' end of the
primer, with the remainder of the primer sequence being substantially
complementary to the priming sequence. Alternatively, non-complementary
bases or modified bases can be interspersed into the extension primer,
provided that base substitutions do not inhibit hybridization.
In addition to the specific priming sequence, the nucleic acid template may
include "non-specific priming sequences" or "nonspecific sequences" to
which the extension primer has varying degrees of complementarity. Thus,
an extension primer may also be capable of hybridizing to non-specific
priming sequences on the template, thereby initiating non-specific priming
events which result in amplification of non-target sequences.
To a certain extent, the greater the sequence complementary between the
extension primer and the priming sequence of the template, the less the
likelihood for non-specific priming events. However, even exact
complementarity between the primer and the priming sequence cannot
eliminate hybridization of the primer to itself, other primers or probes,
or to non-priming sequences on the template to which the primer may also
be complementary.
To substantially reduce the possibility of non-specific priming events, the
invention provides an energy sink oligonucleotide which competes with
non-specific priming sequences for hybridization to the extension primer.
Thus, the term "energy sink oligonucleotide" refers broadly to an
oligonucleotide capable of hybridizing to an extension primer to prevent
non-specific priming events. The energy sink is a molecular entity that is
capable of competitively inhibiting the binding of a primer to nontarget
nucleic acid sequences. The energy sink may be any molecule capable of
performing this function, but preferably is an oligonucleotide of
sufficient complementarity and in sufficient concentration so as to
provide such competitive inhibition. It will be understood by one of
ordinary skill in the art that the degree of complementarity and the
concentration are interdependent variables. Thus, the energy sink may be
perfectly complementary with the primer or alternatively imperfectly
complementary with the primer, provided that the energy sink is present in
sufficient concentration to competitively inhibit binding of the primer to
nontarget sequences. There may even exist a nontarget sequence that is
more complementary with the primer than is the energy sink, again,
provided that the energy sink is present in sufficient concentration to
competitively inhibit binding of the primer to nontarget sequences.
In the preferred embodiments, the energy sink is more complementary to the
primer than to nontarget sequences and there is at least one energy sink
molecule for every primer at the outset of the reaction. Most preferably,
the energy sink is an oligonucleotide having at least ten consecutive
bases perfectly complementary to a ten base sequence of a primer. In one
preferred embodiment, the extension primer and energy sink oligonucleotide
are initially hybridized to each other in a primer:energy sink duplex
prior to initiation of the amplification reaction.
The primer and energy sink need not be fully contiguous, and in certain
preferred embodiments are not. In one such embodiment the 3' terminal end
of the primer protrudes or extends beyond the energy sink when the energy
sink and primer are hybridized. This configuration precludes unintended
extension of the primer in a 5'-3' direction when the primer is hybridized
to the energy sink in a primer:energy sink duplex. In another such
embodiment, the 3' end of the oligonucleotide is noncomplementary with the
5' end of the primer, so as to provide a mechanism for preventing
extension from the 3' end of the energy sink when the energy sink
hybridizes with target nucleic acid. It is necessary only that the primer
and energy sink have sufficient overlapping regions to effect competitive
inhibition as described above.
The energy sink is adapted to prevent initiation by the energy sink, of
nontarget extension products. When the energy sink is an oligonucleotide,
two types of initiation may occur. If the energy sink oligonucleotide
hybridizes to target sequence, then extension may be in a 5' or 3'
direction. If target duplication is in a 5'-3' direction from the primer,
then nontarget extension from the energy sink oligonucleotide would also
be in the 5'-3' direction. To prevent such nontarget extension, the 3'
end of the energy sink oligonucleotide can be modified. Various
modifications are well known to those of ordinary skill in the art and
include 3'-dideoxy, 3'-phosphorylation, 3'-amino termination and the use
of mismatching nucleotides at the 3' end of the energy sink, i.e.,
nucleotides which are not complementary to the target sequence. The second
type of initiation that may occur is that resulting from the energy sink
oligonucleotide hybridizing to nontarget sequences. Again, if conditions
for (preferably) substantially only 5'-3' extension are applied, then the
modifications of the 3' end of the energy sink oligonucleotide as
described above will suffice.
As used herein, the term "primer:energy sink duplex" refers to the complex
formed when the extension primer hybridizes to the energy sink
oligonucleotide. Formation of the complex is a reversible process. Thus,
under appropriate conditions, e.g., an increase in temperature followed by
a reduction in temperature, the duplex may repeatedly dissociate and
reassociate. It is not necessary that the extension primer and energy sink
oligonucleotide have the same length. In one preferred embodiment, the 3'
terminal of the extension product protrudes when the primer is hybridized
to an energy sink oligonucleotide in a primer:energy sink complex.
The energy sink oligonucleotide may also be complementary to and thus
capable of hybridizing to, a sequence in the nucleic acid template.
However, the energy sink oligonucleotide is rendered incapable of acting
as a primer for an extension reaction, such as by removing or modifying
the 3' terminal hydroxy group. Alternatively, a nucleotide may be
incorporated at the 3' terminal position which cannot base pair with a
corresponding nucleotide on the template. These modifications to the 3'
terminal of the energy sink oligonucleotide thus render the energy sink
incapable of acting as a primer of an extension reaction.
As discussed above, the extension primer is capable of binding to (1) a
specific priming sequence, (2) a non-specific priming sequence or (3) an
energy sink oligonucleotide. Assuming that the extension primer is exposed
to the template under conditions favorable to priming and extension, such
as those conditions disclosed in the Examples, at least three different
complexes are formed. The relative proportions of each complex are a
function of concentrations (of the template, the extension primer and the
energy sink oligonucleotide) and the free energy released for
hybridization for each complex. The free energy released for hybridization
of a primer to a complementary sequence to form a nucleic acid complex,
e.g., duplex, may be analogized to the free energy released for a
bimolecular chemical reaction. Accordingly, the magnitude of the free
energy released following hybridization of two nucleic acid strands
indicates whether the colliding nucleic acid strands are more or less
likely to hybridize to one another. Thus, the free energy released
following nucleic acid duplex formation reflects the degree of
complementarity between the hybridizing molecules. The greater the
complementarity between the component strands of the complex, the greater
the free energy released will be for formation of the nucleic acid
complex. Accordingly, complexes having greater degrees of complementarity
will have a higher free energy released for hybridization and their
formation will be favored over that of complexes having a lower free
energy released for hybridization.
Hybridization of the extension primer to a specific priming sequence is
energetically favored because the extension primer is designed to have a
high degree of complementarity with the specific priming sequence.
Hybridization of the extension primer to a nontarget sequence (leading to
amplification of a non-target sequence) is unfavored because the degree of
complementarity between the extension primer and the nontarget sequence
will always be less than that between the extension primer and the energy
sink oligonucleotide. Thus, the present invention provides target
amplification with substantially reduced non-target extension product by
providing an energy sink to eliminate possible hybridization of the
extension primer to nontarget sequences. Because the energy sink is
designed to be complementary to the extension primer, the free energy
released for hybridization of an extension primer to its complementary
oligonucleotide, e.g., energy sink, to form a primer: oligonucleotide
duplex is more than the free energy released for hybridization of that
primer to any nontarget sequence in the template.
Addition of the energy sink oligonucleotide in "sufficient concentration"
to the amplification reaction mixture, prevents amplification of
non-target sequences. As used in reference to the energy sink
oligonucleotide concentration, the term "sufficient concentration" refers
to an amount of energy sink oligonucleotide sufficient to prevent
hybridization of the extension primer to non-specific priming sequences.
Preferably, the energy sink oligonucleotide concentration will be at least
equal to the extension primer concentration. More preferably, the
concentration of the energy sink oligonucleotide will be in excess of 1-2
times the concentration of the extension primer. Preferably, the energy
sink oligonucleotide is initially | | |
