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Description  |
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FIELD OF THE INVENTION
The present invention relates to methods for detecting and measuring
amplification of a nucleic acid target sequence.
BACKGROUND OF THE INVENTION
In vitro nucleic acid amplification techniques have provided powerful tools
for detection and analysis of small amounts of nucleic acids. The extreme
sensitivity of such methods has lead to attempts to develop them for
diagnosis of infectious and genetic diseases, isolation of genes for
analysis, and detection of specific nucleic adds as in forensic medicine.
Nucleic acid amplification techniques can be grouped according to the
temperature requirements of the procedure. The polymerase chain reaction
(PCR; R. K. Saiki, et al. 1985. Science 230, 1350-1354), ligase chain
reaction (LCR; D. Y. Wu, et al. 1989. Genomics 4, 560-569; K. Barfinger,
et al. 1990. Gene 89, 117-122; F. Barany. 1991. Proc. Natl. Acad. Sci. USA
88, 189-193) and transcription-based amplification (D. Y. Kwoh, et al.
1989. Proc. Natl. Acad. Sci. USA 86, 1173-1177) require temperature
cycling. In contrast, methods such as strand displacement amplification
(SDA; G. T. Walker, et al. 1992. Proc. Natl. Acad Sci. USA 89, 392-396 and
G. T. Walker, et al. 1992. Nuc. Acids. Res. 20, 1691-1696, both
disclosures being incorporated herein by reference), self-sustained
sequence replication (3SR; J. C. Guatelli, et al. 1990. Proc. Natl. Acad.
Sci. USA 87, 1874-1878) and the Q.beta. replicase system (P. M. Lizardi,
et al. 1988. BioTechnology 6, 1197-1202) are isothermal reactions. In
addition, WO 90/10064 and WO 91/03573 describe use of the bacteriophage
phi29 replication origin for isothermal replication of nucleic acids.
A variety of methods have also been developed to detect and/or measure
nucleic acid amplification. For the most part, these methods are
primer-based, meaning that they depend on hybridization of a primer to the
target sequence, in some cases followed by extension of the primer.
Primer-based detection of amplified nucleic acids in PCR often relies on
incorporation of an amplification primer into the amplified product
(amplicon) during the amplification reaction. Features engineered into the
PCR amplification primer therefore appear in the amplification product and
can be used either to detect the amplified target sequence or to
immobilize the amplicon for detection by other means. For example,
Syvanen, et al. (1988. Nucleic Acids Res. 16, 11327-11338) report the use
of biotinylated PCR amplification primers to produce biotin-containing
amplification products. These amplicons can then be hybridized to a second
probe containing a fluorescent dye or other reporter group. The hybridized
complex is then selectively isolated from other components of the reaction
mixture by affinity-based immobilization of the biotin-containing complex
and is detected by means of the reporter group. Laongiaru, et al. (1991.
European Patent Application No. 0 420 260) describe a similar use of
biotin-containing PCR amplification primers conjugated to fluorescent dyes
for detection of PCR amplification products. The amplicons containing the
primers are separated from unextended primers on the basis of size, and
multiplex amplification was detected using different fluorescent dyes on
two amplification primer sets. Kemp, et al. (1989. Proc. Natl. Acad. Sci.
USA 86, 2423-2427; 1990. PCT Patent Application No. WO 90/06374) describe
a method for capturing amplified DNA by incorporation of one modified
amplification primer and use of a second modified amplification primer as
a means for detection. The Kemp "capture primer[ contains a 5' tail which
is the single stranded form of the recognition sequence for the
double-stranded DNA binding protein GCN4. The Kemp "detector primer"
includes a biotin moiety on its 5' end. The amplified product is
immobilized by binding to the double-stranded GCN4 recognition sequence
generated by amplification using the capture primer. The biotin moiety
introduced by the detector primer is bound to an avidin-peroxidase complex
to provide colorimetric detection of the immobilized PCR amplification
product. Wahlberg, et al. (1990. Proc. Natl. Acad. Sci. USA 87, 6569-6573)
report a similar method in which one PCR amplification primer is
biotinylated and the other contains a 5' tail encoding the E. coli lac
operator sequence. Double stranded amplification products are immobilized
by binding to streptavidin and detected colorimetrically by binding of a
lac repressor-.beta.-galactosidase fusion protein to the double-stranded
lac operator generated by amplification. The Wahlberg, et al. method
differs from the Kemp, et al. method in that the biotin-streptavidin
interaction rather than the double-stranded binding protein provides
immobilization of the amplification products and the double-stranded
binding protein provides colorimetric detection. This suggests that the
two methods could be combined by using two amplification primers, each
with a 5' tail encoding the recognition sequence of a different
double-stranded binding protein. Amplified products could then be
immobilized by binding to one double stranded binding protein and detected
by binding to the other. C. A. Vary (1992. Clinical Chemistry 38, 687-694;
1992. PCT Patent Application No. WO 92/11390) describes the use of
amplification primers containing 5' tails which form hybridization sites
for a third oligonucleotide when incorporated into otherwise
double-stranded amplicons. Hybridization of one tail was used to capture
the amplified product and the other was used to detect it by hybridization
to a probe conjugated to a fluorescent dye.
All of these primer-based methods of detecting PCR amplification products
require two amplification reactions to achieve high sensitivity, i.e.,
detection of fewer than 100 copies of the target sequence. That is, a
first amplification of the target sequence is followed by a second
amplification using nested primers incorporating the desired modifications
for capture and/or detection. Two consecutive amplifications in this
manner are needed to avoid unacceptably high levels of background signal
produced by amplification of non-target DNA spuriously primed with the
modified, signal-generating primers. This feature of the prior art methods
makes them time-consuming and cumbersome, and the advantages of
primer-based detection methods are therefore often offset by the
requirement for a second consecutive amplification reaction.
Non-specific amplification of DNA would be expected to present particular
problems for primer-based detection of amplification products in SDA
reactions because these amplifications are carried out at a relatively low
temperature (about 37.degree.-40.degree. C.) which would allow increased
mispriming as compared to PCR, resulting in even higher levels of
background signal. Unexpectedly, the instant methods for primer-based
detection of SDA resulted in low levels of background signal in spite of
the use of only a single amplification reaction which generates products
for detection concurrently with amplification of the target sequence.
Simultaneous or concurrent generation of a secondary amplification product
and the amplified target sequence is referred to herein as real-time
primer extension, real-time detection of amplification, etc.
SUMMARY OF THE INVENTION
The instant invention provides methods for detecting, immobilizing
(capturing) or localizing primer extension products of an SDA reaction
which are coupled to, and an indication of, amplification of the target
sequence. The primer extension products are secondary, target-specific DNA
products generated during SDA of the target sequence and can therefore be
used to detect and/or measure target sequence amplification. The secondary
products, however, are not amplifiable and remain inert in the SDA
reaction after they are formed without interfering with the exponential
amplification of the target sequence. The secondary product can be
designed or modified to contain special features to facilitate its
detection, immobilization (capture) or localization. The inventive methods
are useful for real-time monitoring of SDA reactions, especially in
situations where detection of target sequence amplicons would interfere
with further amplification or manipulation. The instant methods will also
be useful for detection of amplification products in fixed cells after in
situ SDA, especially when the secondary products contain 5' tail sequences
to facilitate detection or localization of amplification products.
DESCRIPTION OF THE DRAWINGS
FIG. 1A and FIG. 1B illustrate the steps of the methods of the invention.
FIG. 1A illustrates the production of the secondary amplification product
from a single stranded target sequence using two signal primers. FIG. 1B
shows the analogous process originating from the complementary strand when
the original target sequence is double stranded.
FIG. 2 illustrates the production of the secondary product using a single
signal primer.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a method for detecting, monitoring or localizing
amplification products of SDA reactions by real-time primer extension.
Amplification of a target sequence by SDA is detected, monitored or
localized by simultaneously generating a secondary amplification product,
the production of which is tightly coupled to amplification of the target
sequence. This secondary amplification product is produced during the SDA
reaction without requiring any additional additional amplification steps
or manipulations. Once generated, the secondary amplification product is
inert in the reaction mixture and does not interfere with or inhibit
normal SDA of the desired target sequence. The methods are therefore
useful for real-time monitoring of SDA and detecting amplification of the
target sequence, especially in situations where detection of the amplified
target sequence itself would inhibit or prevent further reaction or
manipulation of the amplicons.
The present invention provides a primer-based amplification detection
method in which the need for a second amplification reaction is
eliminated. The method employs signal primers which are similar to capture
and detector probes and do not function as amplification primers in the
SDA reaction. Consequently, any extension products formed through errant
extension of these signal primers on non-target templates cannot undergo
subsequent amplification. Because mispriming itself is comparatively rare,
it is detectable only after subsequent amplification of the misprimed
sequence. In the absence of such subsequent amplification, as in the
methods of the present invention, the signal primers may be added to the
amplification reaction prior to initiation of amplification with no
apparent increase in background signal levels. This greatly simplifies the
detection procedure and makes possible homogeneous real-time analysis of
SDA reactions.
As used herein, the following terms and phrases are defined as follows:
An amplification primer is a primer for amplification of a target sequence
by primer extension. For SDA, the 3' end of the amplification primer (the
target binding sequence) hybridizes at the 3' end of the target sequence.
The amplification primer comprises a recognition site for a restriction
endonuclease near its 5' end. The recognition site is for a restriction
endonuclease which will cleave one strand of a DNA duplex when the
recognition site is hemimodified ("nicking"), as described by Walker, et
al. (1992. PNAS, supra). A hemimodified recognition site is a double
stranded recognition site for a restriction endonuclease in which one
strand contains at least one derivatized nucleotide which causes the
restriction endonuclease to nick the primer strand rather than cleave both
strands of the recognition site. Usually, the primer strand of the
hemimodified recognition site does not contain derivatized nucleotides and
is nicked by the restriction endonuclease. Alternatively, the primer may
contain derivatized nucleotides which cause the unmodified target strand
to be protected from cleavage while the modified primer strand is nicked.
The preferred hemimodified recognition sites are hemiphosphorothioated
recognition sites for the restriction endonucleases HincII, HindII, AvaI,
NciI and Fnu4HI. The amplification primer also comprises a 3'-OH group
which is extendable by DNA polymerase when the target binding sequence of
the amplification primer is hybridized to the target sequence. For the
majority of the SDA reaction, the amplification primer is responsible for
exponential amplification of the target sequence.
Extension products are nucleic acids which comprise a primer and a newly
synthesized strand which is the complement of the target sequence
downstream of the primer binding site. Extension products result from
hybridization of a primer to a target sequence and extension of the primer
by polymerase using the target sequence as a template.
A bumper primer is a primer which anneals to a target sequence upstream of
the amplification primer, such that extension of the bumper primer
displaces the downstream amplification primer and its extension product.
Extension of bumper primers is one method for displacing the extension
products of amplification primers, but heating is also suitable.
Identical sequences will hybridize to the same complementary nucleotide
sequence. Substantially identical sequences are sufficiently similar in
their nucleotide sequence that they also hybridize to the same partially
complementary nucleotide sequence.
The terms target or target sequence refer to nucleic acid sequences to be
amplified. These include the original nucleic acid sequence to be
amplified, its complementary second strand and either strand of a copy of
the original sequence which is produced in the amplification reaction. The
target sequence may also be referred to as a template for extension of
hybridized amplification primers.
A signal primer is a primer which hybridizes to a target sequence
downstream of an amplification primer such that extension of the
amplification primer displaces the signal primer and its extension
product. The signal primer comprises a 3'-OH group which can be extended
by DNA polymerase when the signal primer is hybridized to the target
sequence. The signal primer may be unmodified, e.g., for detection of
secondary amplification products based on their size. Alternatively, the
signal primer may include a reporter group or label, or a structural
feature to facilitate detection of its extension product.
Amplification products, amplified products or amplicons are copies of the
target sequence generated by hybridization and extension of an
amplification primer. This term refers to both single stranded and double
stranded amplification primer extension products which contain a copy of
the original target sequence, including intermediates of the amplification
reaction.
Secondary amplification products or secondary products are copies of the
target sequence generated by hybridization and extension of a signal
primer. The secondary amplification product comprises an internal segment
of the amplified target sequence. These terms also refer to both single
stranded and double stranded extension products of signal primers,
including intermediates in the process which generates the final double
stranded form. In contrast to amplification products, the double stranded
secondary amplification product is generally not available for further
amplification, although some secondary amplification products may be
amplifiable in a linear fashion.
In the methods of the invention, amplification primers for SDA are
hybridized to a target sequence and the target sequence is amplified
generally as described by Walker, et al., 1993 PNAS or Walker, et al. 1993
Nuc. Acids Res., supra. As described in these two publications, the target
sequence may be prepared for SDA either by restricting total DNA with an
appropriate restriction endonuclease (e.g., HincII) or by generating
target fragments having the appropriate restriction endonuclease
recognition sites at the ends using bumper primers and amplification
primers. Prepared fragments containing the target sequence are then
amplified by SDA as described. However, the SDA reaction of the invention
further comprises at least one signal primer which results in simultaneous
or concurrent generation of a secondary amplification product for use in
detecting, monitoring or localizing amplification products produced by the
SDA reaction. The secondary amplification products may also contain
features which facilitate their capture or immobilization, so that they
may be isolated for detection, quantitation or further manipulation. The
secondary amplification products are produced in the SDA reaction by
inclusion of at least one signal primer in the reaction mixture. For
certain applications, it may be preferable to include a pair of signal
primers. The signal primer or signal primers hybridize to the target
sequence downstream of the hybridization site of the amplification
primers. They are extended by polymerase in a manner similar to extension
of the amplification primers. The signal primer hybridizes at a site in
the target sequence such that extension of the amplification primer
displaces the extension product of the signal primer. At least the 3' end
of the signal primer comprises a sequence which hybridizes to the target
sequence. The entire signal primer may hybridize to the target sequence,
for example when it is unmodified or chemically modified for detection by
addition of a reporter group, label or affinity ligand. Alternatively, the
5' end of the signal primer may comprise a sequence which does not
hybridize to the target sequence but which contains special nucleotide
sequences (often involving structural features) which facilitate detection
or capture of the secondary amplification product. These chemical
modifications and special sequences are incorporated into the secondary
amplification products when the signal primers are hybridized and extended
on a template. Examples of chemical modifications include affinity ligands
(e.g., avidin, streptavidin, biotin, haptens, antigens and antibodies) and
reporter groups (labels, e.g., radioisotopes, fluorescent dyes, enzymes
which react to produce detectable reaction products, and visible dyes).
Examples of special nucleotide sequences include (i) sequences which will
form a triple helix by hybridization of a labeled oligonucleotide probe to
the double stranded secondary amplification product, and (ii) recognition
sites for double-stranded DNA binding proteins which become capable of
binding the double-stranded DNA binding protein when rendered double
stranded during amplification (e.g., repressors, regulatory proteins,
restriction endonucleases, RNA polymerase). Nucleotide sequences which
result in double stranded restriction endonuclease recognition sites are a
preferred structural feature for use in signal primers, as subsequent
restriction may be used to generate a secondary amplification product
which is recognizable by a characteristic size.
When the inventive methods employ two signal primers which hybridize to
opposite strands of a double stranded target sequence, as illustrated in
FIGS. 1A and 1B, one of the signal primers may contain a special
nucleotide sequence or chemical modification to facilitate capture or
immobilization of the secondary amplification product and the other may
contain a detectable reporter group or label for detection of the captured
or immobilized secondary amplification product. The use of labels and
reporter groups for detecting nucleic acids as well as the use of ligands,
chemical modifications and nucleic acid structural features for capture or
immobilization of nucleic acids is well known in the art. Alternatively,
the signal primer may be unmodified, i.e., without reporter groups,
capture groups or structural features to facilitate detection or capture
of the secondary amplification products. The secondary amplification
products may then be detected based on their size, e.g., by gel
electrophoresis and ethidium bromide staining. All of these methods are
useful in the present invention and one skilled in the art can routinely
select appropriate methods for use in any particular amplification assay
system.
It is an important feature of the invention that the signal primers do not
function as amplification primers in the SDA reaction in which they are
employed. Without wishing to be bound by any specific mechanism by which
the inventive methods work, Applicants believe it is this feature which
allows the signal primers to be added to the amplification reaction
mixture without promoting the high levels of background signal generated
by other primer-based methods. High levels of background signal are
believed to be due to non-specific priming and subsequent amplification of
spuriously primed non-target DNA when the primers are capable of
functioning as amplification primers. The present invention therefore
greatly simplifies the procedures for primer-based detection methods,
which previously relied on two consecutive amplification reactions to
attain high sensitivity and specificity, the second reaction being
performed with internally nested signal-generating amplification primers.
As stated above, nucleic acid fragments having appropriate restriction
endonuclease recognition sequences at the ends and containing the target
sequence may be prepared for amplification either as described by Walker,
et al. 1992. PNAS, supra or as described by Walker, et al. 1992 Nuc. Acids
Res., supra. For simplicity, the illustrations of the inventive methods in
FIG. 1A, FIG. 1B and FIG. 2 begin with a nucleic acid fragment containing
the target sequence. If prepared according to Walker, et al. 1992. PNAS,
supra, it represents restricted double stranded DNA which has been
denatured. If prepared according to Walker, et al. 1992. Nuc. Acids Res.,
supra, appropriate restriction endonuclease recognition sites are added to
the fragment according to the disclosed target generation scheme. It is
believed that bumper, amplification and signal primers may simultaneously
hybridize to a target sequence in the target generation scheme of Walker,
et al. (1992. Nuc. Acids Res., supra), extension of each upstream primer
displacing the extension product of the downstream primer and
simultaneously generating amplifiable target fragments and secondary
amplification products.
FIG. 1A and FIG. 1B illustrate one embodiment of the invention in which a
pair of signal primers are used for detecting amplification of a
double-stranded target sequence (5'-A-B-C-D/5'-D'-C'-B'-A'). FIG. 1A
illustrates the method of the invention for the first of the two
complementary strands of the target sequence (5'-D'-C'-B'-A'). The raised
portion of the amplification primers illustrated in FIGS. 1A, 1B and 2
indicates a nickable restriction endonuclease recognition site as
described above and by Walker, et al. (1992. PNAS and Nuc. Acids Res.).
Long raised portions illustrate full-length restriction endonuclease
recognition sites and short raised portions illustrate partial restriction
endonuclease recognition sites, generally produced alter nicking and
displacing a strand. The nucleic acid fragments comprising the target
sequence may be generated either by endonuclease restriction of larger
nucleic acids (Walker, et al. 1992. PNAS, supra) or by target generation
as described by Walker, et al. (1992. Nuc. Acids Res., supra). However,
for purposes of illustration and to simplify the diagrams, FIGS. 1A, 1B
and 2 begin with the target sequence contained on a nucleic acid fragment
previously restricted with a restriction endonuclease which does not cut
the target sequence.
In FIG. 1A, signal primer R-B is included in the SDA reaction mixture and
hybridizes to the target sequence downstream of a first amplification
primer by hybridization of the B portion of the signal primer to B'. The R
portion of the R-B signal primer sequence includes a reporter group or
label, or is a structural feature to facilitate detection or capture. R
may or may not hybridize, as discussed above, but is shown here as not
hybridizing to clarify the different functional features of the signal
primer. For the purposes of this illustration, R will contain a reporter
group, but may contain other chemical modifications or structural features
as discussed above. Both amplification primer A and signal primer R-B are
extended by DNA polymerase using the target sequence as a template. The
signal primer extension product R-B-C-D (structure #1) is displaced from
the template by extension of amplification primer A and in turn serves as
a template for hybridization and extension of a second signal primer Q'-C'
and a second amplification primer D'. The C' portion of the Q'-C' sequence
hybridizes to C. The Q' portion of the second signal primer is analogous
to R, and for purposes of this illustration Q' will contain a modification
or sequence to facilitate capture of the secondary amplification product.
The Q'-C' extension product is displaced by extension of the second
amplification primer. The displaced Q'-C' extension product (structure #2)
then serves as a template for hybridization and extension of R-B,
resulting in a double stranded, target-specific secondary amplification
product (structure #3) which comprises the terminal segments (R and Q') of
the signal primers and the internal segment B'-C' of the target sequence.
As the secondary amplification product does not contain nickable
restriction endonuclease recognition sites, it is not amplifiable in the
SDA reaction and remains effectively inert throughout the remainder of the
amplification reaction, but additional copies of the secondary
amplification product are generated from the target sequence.
Hybridization and extension of the second amplification primer (D'), in
addition to displacing the R'-B'-C'-Q' extension product, generates a
double stranded fragment with the R/R' sequence at one end and a
hemimodified restriction endonuclease recognition site at the other end
(structure #4). This restriction endonuclease recognition site is nickable
by the restriction endonuclease present in the SDA reaction. The DNA
polymerase present in the SDA reaction can then initiate polymerization
and displacement at the nick, resulting in the illustrated R'-B'-C'-D'
product comprising a portion of the restriction endonuclease recognition
site. This product can be made double-stranded by hybridization and
extension of R-B (structure #5). Although cyclically repeating the
nicking, polymerizing and displacing cycle amplifies this fragment at a
linear rate, generally neither the single-stranded or double-stranded
product will be detectable by virtue of the absence of the Q/Q' portion
containing the modification or sequence to facilitate capture. If the
functions of Q/Q' and R/R' are reversed, (i.e., Q/Q' contains the reporter
group or label and R/R' contains the modification or sequence to
facilitate capture), these products, though captured, would not be
detectable by virtue of the absence of the reporter group or label. It
should be understood, however, that structure #5 may be detectable when
the reporter group is detectable independent of capture, e.g., when the
reporter group is a fluorescent label detectable by anisotropy or
fluorescence polarization (WO 92/18650; R. Devlin, et al. 1993. Clin.
Chem. 39, 1939-1943) or a radioisotope which can be detected by gel
electrophoresis and autoradiography.
FIG. 1A also shows how extension of the first amplification primer on the
target sequence, in addition to displacing the extension product of R-B,
generates the double-stranded target sequence with the hemimodified,
nickable restriction endonuclease recognition site which is required for
amplification of the target sequence by SDA (structure #6). These reaction
products enter the conventional SDA reaction and are amplified. Formation
of the secondary amplification product is therefore tightly coupled to
amplification of the target sequence and is useful to monitor whether or
not amplification has taken place as well as to provide a measure of
target amplification. In spite of the tight linkage of generation of the
secondary amplification product and generation of amplification products,
however, amplification of the target sequence is not inhibited provided
essential reaction components are present in excess. In addition,
amplified target sequences may also bind signal primers, resulting in
generation of additional copies of the secondary amplification products.
FIG. 1B illustrates generation of secondary amplification products from the
complementary second strand of the double-stranded target sequence
(5'-A-B-C-D). In general, the reaction steps for the complementary second
strand are similar to those for the first strand. However, the second
amplification primer (D') and signal primer C'-Q' hybridize first to the
complementary strand and are extended. The first amplification primer (A)
and signal primer R-B then hybridize to the displaced extension product of
C'-Q' (A'-B'-C'-Q', structure #7) and are extended to produce R-B-C-Q
(structure #8). Hybridization of Q'-C' to R-B-C-Q and extension results in
the double stranded secondary amplification product R'-B'-C'-Q'/R-B-C-Q
(structure #9). This secondary amplification product is detectable in
systems requiring both capture and reporter groups due to the presence of
both features in structure #9. The reaction for the complementary strand
also produces a reaction product which can be linearly amplified by
nicking, polymerizing and displacing (structure #10). The displaced single
strand of this linear amplification becomes double stranded by
hybridization and extension of Q'-C' (structure #11 ). Generally, neither
the single or double stranded reaction products of this linear
amplification are detectable due to the absence of either the reporter
group or the capture group. They are not further amplifiable because they
lack an intact restriction endonuclease recognition site. However, if Q
comprises a reporter group which is detectable independent of capture
(e.g., a fluorescent label or a radioisotope as described above),
structures #10 and #11 will also be detectable.
Detection specificity will generally be improved when two signal primers
are employed as in FIG. 1A and FIG. 1B, but a single signal primer may
also be used. This method is illustrated in FIG. 2. In this case, the
signal primer may contain either a capture group or a reporter group, and
the target sequence itself or an amplification primer may optionally
provide a second capture or reporter group. Alternatively, when both a
capture and reporter group are required, the signal primer may contain
both a capture and a reporter group which act in conjunction only when the
signal oligonucleotide becomes double-stranded. This structure is formed
only when the presence of target sequences induces priming, extension,
displacement and re-priming as shown in FIG. 2. Such bi-functional signal
primers may also form the basis for a variety of homogeneous detection
methods such as fluorescence anisotropy or fluorescence energy transfer.
To generate secondary amplification products using a single signal primer
according to FIG. 2, a first amplification primer (A) and the signal
primer R-B are hybridized to a single stranded target sequence
A'-B'-C'-D'. Both primers are extended, and extension of the first
amplification primer displaces the extension product of signal primer R-B
(R-B-C-D), producing structure #1. As there is no second signal primer,
only the second amplification primer (D') hybridizes to P-B-C-D and is
extended, generating structure #2 with a nickable, hemimodified
restriction endonuclease recognition site. Linear amplification of this
product by nicking, polymerizing and displacing, as shown, generates
fragments to which the signal primer can hybridize and be extended. This
generates the double stranded secondary amplification product, structure
#3. It is not amplifiable due to the lack of an intact restriction
endonuclease recognition site, but is detectable by virtue of R/R' when
the reporter or capture group is detectable only in double stranded form
or by virtue of R when the reporter group is detectable alone. When
detection of the reporter group does not require double-strandedness
(e.g., a fluorescent label), structures #1, #2 and #3 are detectable as
secondary amplification products.
EXAMPLE 1
The real-time detection of amplification of the instant invention was
compared to conventional post-amplification detection of amplified target
sequences. Fragments of the IS6110 sequence of Mycobacterium tuberculosis
(M.tb) were amplified in SDA reactions performed essentially as described
by Walker, et al. (1992. Nuc. Acids Res.), except that each 60 .mu.L
reaction mixture contained 0.2 .mu.g of human placental DNA and varying
amounts of genomic M.tb DNA. Amplification primer sequences (S.sub.1 and
S.sub.2) and bumper primer sequences (B.sub.1 and B.sub.2) were also as in
Walker, et al. (1992. Nuc. Acids Res.) For the amplification reactions
incorporating signal primers, the .sup.32 P-labeled signal primer .sup.32
P-CGTTATCCACCATAC (SEQ ID NO:1) was added to the reactions prior to
amplification at a final concentration of 60 nM. Predicted secondary
amplification products produced in these reactions were 35 and 56
nucleotides in length. For post-amplification detection of amplified
target sequences, one-tenth of the reaction mixture was used to detect
amplification products by primer extension of SEQ ID NO:1 as described by
Walker, et al. (1992. Nucl. Acids. Res., supra), producing extension
products either 35 or 56 nucleotides in length.
Amplification was allowed to proceed for 2 hr. at 37.degree. C. in the
presence of 1 to 500,000 genome copies of M.tb. After stopping the
amplification, one-tenth of each reaction was subjected to electrophoresis
on denaturing polyacrylamide gels. As little as one copy of M.tb genomic
DNA was detected using the signal primer according to the invention. Also,
the signal intensity decreased with decreasing target levels, indicating
that the levels of secondary amplification product reflect the degree of
target sequence amplification. The real-time extension of the signal
primer appeared on the gel to be several fold less sensitive than the
conventional post-amplification primer extension method, possibly because
the .sup.32 P-labeled signal primer was present during SDA at
concentrations about 10-fold less than the SDA primers. If SDA primers are
extended on the target sequence before a signal primer binds and is
extended, no signal will result. Thus, higher concentrations of signal
primer should increase the method's sensitivity by improving hybridization
kinetics for the signal primer. Higher signal primer concentrations are
therefore preferred when reaction products are separated for detection,
but the concentration of amplification primers may be kept similar to the
concentrations used in conventional SDA. However, lower signal primer
concentrations are preferred to keep background low for homogeneous
detection methods such as fluorescence anisotropy. The lower
concentrations of signal primer are preferably used with lower
concentrations of polymerase and the amplification primer which hybridizes
upstream of the signal primer than is customary in conventional SDA. This
experiment also demonstrated that the presence of the signal primer in the
amplification reaction mixture does not lead to significant levels of
background signal. In fact, background signal levels appeared to be lower
in samples detected by real-time signal-primer extension as compared to
post-amplification primer extension.
EXAMPLE 2
SDA reactions were performed generally as previously described (Walker.
1993. PCR--Methods and Applications 3, 1) in 50 mM KiPO.sub.4 (pH 7.5),
0.1 mg/mL bovine serum albumin, 0.5 mM dUTP, 0.2 mM each dGTP, dCTP and
dATP.alpha.S, 7 mM MgCl.sub.2, 11% (v/v) glycerol, the indicated
concentrations of amplification primers, 25 nM bumper primers, 50 ng human
placental DNA, the indicated amount of exonuclease deficient Klenow
(United States Biochemicals), 150 units HincII (New England Biolabs).
Reactions were run for the indicated time at 41.degree. C. SDA reactions
contained varying amounts of M.tb DNA, which contains the IS6110 target
sequence for amplification. The S.sub.1 amplification primer sequence, the
B.sub.1 bumper primer sequence and the B.sub.2 bumper primer sequence used
were as described by Walker, et al. (1992. Nuc. Acids Res., supra). The
S.sub.2 amplification primer (SEQ ID NO:2) had the target binding sequence
and HincII site disclosed by these authors, but comprised a different
sequence at the 5' end. The amplification primers hybridize to nucleotide
positions 972-984 and 1011-1023 of the IS6110 sequence. The bumper primers
hybridize to nucleotide positions 954-966 and 1032-1044. Secondary
amplification products were visualized by autoradiography after
electrophoresis on denaturing polyacrylamide gels.
SDA reactions were performed for 3 hrs. in the presence of 0.1 nM of a
5'-.sup.32 P-labeled signal primer (SEQ ID NO:3). This signal primer is 28
nucleotides in length and hybridizes to nucleotide positions 985-1012 of
the IS6110 target sequence, between the amplification primers. S.sub.1 and
S.sub.2 were present at 180 and 30 nM, respectively. Exonuclease deficient
Klenow was used at 0.25 units. Samples 1-4 contained 100, 10, 1 and 0 M.tb
genome molecules, respectively. During the SDA reaction, SEQ ID NO:3 is
extended by polymerase to a length of 44 nucleotides using the target
sequence as a template. As discussed above, this template is most likely
primarily the displaced, amplified target strand generated during SDA, but
concurrent extension of the bumper, amplification and sig | | |
