Detection of hydrophobic amplification products by extraction into an organic phase

Amplification of a target sequence is detected qualitatively or quantitatively by concurrent generation of secondary amplification products labeled with a lipophilic label. The secondary amplification products are designed such that they are generated and cleaved or nicked in a target amplification-dependent manner. This reduces the number of hydrophilic nucleotides linked to the lipophilic label and allows the cleaved or nicked secondary amplification product comprising the lipophilic label to be transferred from the aqueous reaction phase to an organic phase for detection as an indicator of target amplification.

FIELD OF THE INVENTION 
The invention relates to detection of nucleic acid amplification and in 
particular to detection of nucleic acid amplification by concurrent 
generation of secondary amplification products. 
BACKGROUND OF THE INVENTION 
In vitro nucleic acid amplification techniques provide 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 acids 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. Barringer, 
et al. 1990. Gene 89, 117-122; F. Barany. 1991. Proc. Natl. Acad. Sci. 
U.S.A. 88, 189-193) and transcription-based amplification (D. Y. Kwoh, et 
al. 1989. Proc. Natl. Acad. Sci. U.S.A. 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. U.S.A. 89, 
392-396; G. T. Walker, et al. 1992. Nuc. Acids. Res. 20, 1691-1696; U.S. 
Pat. No. 5,270,184), self-sustained sequence replication (3SR; J. C. 
Guatelli, et al. 1990. Proc. Natl. Acad. Sci. U.S.A. 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 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. However, 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 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. 
P. M. Holland, et al. (1992. Clin. Chem. 38, 462-463) describe a method for 
detecting amplification products of PCR in which the 5'-3' exonuclease 
activity of Taq DNA polymerase is used to generate target 
amplification-specific signal by digestion of a labeled probe hybridized 
downstream of the amplification primer. The labeled probe is not 
extendable, possibly because certain of the detection systems described 
make use of a 3' end-label. Further, an extendable labeled probe would 
function as a PCR amplification primer, thereby increasing non-specific 
background signal in the reaction. Cleaved probe fragments are generated 
during amplification, and may be differentiated from uncleaved probe in a 
variety of ways, depending on the type of probe label. The authors suggest 
thin-layer chromatography or capture by a 3' biotin label to separate 
cleaved from uncleaved probe, or sequencing. These detection methods 
require cumbersome and time consuming manipulations of the sample after 
amplification. The present methods for primer-based detection of target 
amplification also make use of a single amplification reaction to 
concurrently generate secondary products for detection. In contrast to P. 
M. Holland, et al. and other prior art methods, however, the secondary 
amplification products are detected in a simple format by extraction into 
an organic phase. 
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. 
Such restriction endonucleases can be identified in routine screening 
systems in which a derivatized dNTP is incorporated into a restriction 
endonuclease recognition site for the enzyme. The preferred hemimodified 
recognition sites are hemiphosphorothioated recognition sites for the 
restriction endonucleases HincII, HindII, AvaI, NciI, Fnu4HI, BsoBI and 
BsrI. 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. As no special sequences 
or structures are required, amplification primers for PCR generally 
consist only of target binding sequences. 
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. 
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 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, a portion of the signal 
primer or the signal primer extension product. The signal primer further 
comprises a lipophilic reporter group or label which facilitates detection 
of secondary amplification products generated from the signal primer. 
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 oligonucleotides 
generated from a signal primer in a target amplification-dependent manner. 
These terms refer to single stranded or double stranded products generated 
from signal primers, as well as portions of signal primers or signal 
primer extension products generated as a result of target amplification. 
Cleavage of an oligonucleotide refers to breaking the phosphodiester bonds 
of the molecule such that two oligonucleotide cleavage products are 
produced, i.e., breaking the bonds of both strands of a DNA duplex or 
breaking the bond of single-stranded DNA. This is in contrast to nicking, 
which refers to breaking the phosphodiester bond of only one of the two 
strands in a DNA duplex. 
SUMMARY OF THE INVENTION 
The present invention provides methods for detecting amplification of a 
target sequence. Secondary, target-specific amplification products are 
generated from signal primers in a reaction which is coupled to target 
amplification. The secondary amplification products can therefore be used 
to detect and/or measure target sequence amplification. The secondary 
amplification product is an oligonucleotide which comprises a lipophilic 
label. The labeled secondary amplification product is designed such that 
it is cleaved or nicked in a target amplification-dependent manner during 
amplification, thereby reducing the number of hydrophilic nucleotides 
linked to the lipophilic label and allowing the labeled cleavage product 
to be transferred from the aqueous phase of the amplification reaction 
into an organic phase. The more hydrophilic uncleaved or unnicked signal 
primer remains in the aqueous phase as a result of the greater number of 
hydrophilic nucleotides linked to the lipophilic label. The label 
transferred to the organic phase is detected as an indicator of target 
amplification. Detection of the label may be a qualitative or quantitative 
measure of amplification of a target sequence. The inventive methods are 
especially useful for monitoring amplification reactions in situations 
where direct detection of target sequence amplicons would interfere with 
further manipulations or procedures. Organic extraction for detection of 
the secondary amplification products provides a highly sensitive detection 
system in a procedurally simple and rapid format.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is a method for detecting or monitoring amplification 
of a target by conversion of a hydrophilic signal primer comprising a 
lipophilic label to a lipophilic secondary amplification product which is 
transferred from the aqueous reaction phase to an organic phase for 
detection. Conversion takes place in a target amplification-dependent 
manner. That is, production of the secondary amplification product is 
coupled to amplification of the target sequence. Further, the secondary 
amplification product is generated concurrently with amplification of the 
target sequence. Once generated, the secondary amplification product does 
not interfere with or inhibit normal amplification of the desired target 
sequence. The methods are therefore especially useful for detecting 
amplification of the target sequence in situations where direct detection 
of the amplified target sequence itself would inhibit or prevent further 
reaction or manipulation of the amplicons. The lipophilic properties of 
the secondary amplification product allow simple separation from 
unreacted, labeled signal primers, e.g., by transfer to an organic phase. 
In one embodiment of the invention, amplification primers for SDA are 
hybridized to a target sequence which is then amplified generally as 
described by Walker, et al. (1992. PNAS or 1992 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., RsaI) 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. 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 or monitoring amplification of 
the target sequence. For certain applications, it may be preferable to 
include a pair of signal primers, each of which hybridizes to one of the 
two strands of a double-stranded target sequence. The signal primer 
hybridizes to the target sequence downstream of the hybridization site of 
the amplification primer. It may be extended by polymerase in a manner 
similar to extension of the amplification primers, but this feature is not 
required in all amplification systems (for example the PCR method of 
Example 4). That is, the signal primer hybridizes at a site within the 
target sequence such that extension of the amplification primer displaces 
the signal primer, a portion of the signal primer or the signal primer 
extension product. At least the 3' end of the signal primer comprises a 
sequence which hybridizes to the target sequence. Alternatively, the 
entire signal primer may hybridize to the target sequence, depending on 
the method selected for reducing the number of nucleotides in the signal 
primer The signal pruner is further labeled at its 5' end with a 
lipophilic label. The lipophilic label becomes incorporated into the 
secondary amplification product as a result of target amplification. In 
this embodiment, the signal primers cannot function as amplification 
primers in the SDA reaction because they lack a nickable restriction 
endonuclease recognition site. Consequently, any extension products formed 
through errant extension of a signal primer on a non-target template 
cannot undergo subsequent amplification. Because mispriming itself is 
comparatively rare, it is generally detectable only after subsequent 
amplification of the misprimed sequence. Therefore, in the absence of 
subsequent amplification, the signal primers may be present in the 
amplification reaction with no significant increase in background signal 
levels This, in conjunction with simple organic extraction to separate 
labeled secondary amplification products for analysis, greatly simplifies 
the procedure. 
Generation of a double-stranded secondary amplification product during SDA 
is illustrated in FIG. 1. The top portion of FIG. 1 illustrates the 
amplification reaction occuring in SDA. As stated above, nucleic acid 
fragments having appropriate restriction endonuclease recognition 
sequences at the ends and containing the target sequence may be prepared 
for SDA 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 in FIGS. 1 and 2 begin with an amplifiable 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. During SDA target generation, the bumper, amplification 
and signal primers hybridize to a target sequence in the target generation 
scheme, with extension of each upstream primer displacing the downstream 
primer and concurrently generating amplifiable target fragments and 
secondary amplification products. 
The bottom portion of FIG. 1 illustrates the concurrent conversion of the 
single-stranded signal primer to double-stranded form. A signal primer 
hybridizes to one strand of the target sequence downstream of an 
amplification primer (I). Both the amplification primer and the signal 
primer are extended by DNA polymerase using the target sequence as a 
template (II). The signal primer extension product is displaced from the 
template by extension of the amplification primer and in turn serves as a 
template for hybridization and extension of a second amplification primer 
(III and IV), rendering the signal primer extension product 
double-stranded (V). A second signal primer which hybridizes to the second 
strand of a double stranded target sequence may optionally be included in 
the reaction. The second signal primer hybridizes to the second strand of 
the target sequence downstream of the second amplification primer and is 
extended and displaced by extension of the second amplification primer. 
The second signal primer extension product is rendered double stranded by 
hybridization and extension of the first amplification primer. It will 
also be apparent from the illustration of the invention in FIG. 1 that 
multiple signal primers may be employed, each hybridizing to the target 
sequence downstream of the other on the same strand, and all signal 
primers being hybridized downstream of the amplification primer. In this 
manner, each signal primer is displaced by extension of the upstream 
signal primer and the most 5' signal primer is displaced by the 
amplification primer. The multiple signal primers should be designed so 
that they do not hybridize to each other or to the amplification primer 
for the opposite strand of the target sequence. Use of multiple signal 
primers has the advantage of increasing or amplifying the signal generated 
per target, with an increase in sensitivity of the assay, 
The number of nucleotides linked to the lipophilic label in the double 
stranded secondary amplification product is reduced in a target 
amplification-dependent manner to facilitate organic extraction and 
detection of the label. That is, unreacted, single stranded signal primer 
molecules with the linked label are prevented from entering the organic 
phase by their greater number of linked nucleotides, which give the 
molecule more hydrophilic properties. In one embodiment, the signal primer 
comprises a restriction endonuclease recognition site placed such that 
cleavage or nicking of the double-stranded secondary amplification product 
by the restriction endonuclease generates a labeled fragment comprising a 
reduced number of nucleotides which is sufficiently lipophilic to be 
transferred from the aqueous amplification reaction to an organic phase. 
This recognition site should be for a restriction endonuclease which does 
not cleave the target sequence. Typically, such a restriction endonuclease 
recognition site will be placed toward the 5' end of the signal primer to 
minimize the number of nucleotides remaining linked to the label after 
cleavage or nicking. The restriction endonuclease does not cleave its 
recognition site in the single stranded signal primer. However, the signal 
primer becomes cleavable or nickable by the restriction endonuclease when 
convened from single- to double-stranded form during target amplification. 
Therefore, only double-stranded secondary amplification products produced 
during target amplification will be cleaved or nicked and transferred to 
the organic phase. 
The reaction in which a restriction endonuclease recognition site is 
cleaved is illustrated in FIG. 2, where the restriction endonuclease 
recognition site is shown as a raised portion on the nucleic acid strand. 
The initial steps for generation of the double-stranded secondary 
amplification product from the single-stranded signal primer (structures 
I-V) are those illustrated in FIG. 1 (I-V). FIG. 2 illustrates a 
double-stranded restriction endonuclease recognition site in structure V 
(raised blocks) which is cleavable by the restriction endonuclease to 
generate two cleavage products--a lipophilic cleavage product and an 
unlabeled oligonucleotide cleavage product. In addition, the restriction 
endonuclease recognition site at the 5' end of the extended S.sub.2 primer 
in structure V is nickable and can be displaced, producing a strand to 
which the signal primer can hybridize (VI). The double stranded 
restriction endonuclease recognition site in VI may also be cleaved to 
generate a labeled lipophilic cleavage product (VI'), or it may be 
extended by polymerase to generate a fully double-stranded molecule (VII) 
prior to cleavage and generation of the labeled lipophilic cleavage 
product (VII'). V', VI' and VII' represent alternative reaction pathways 
for generation of labeled lipophilic secondary amplification products. As 
illustrated, the recognition site becomes double stranded during target 
amplification and is cleaved by the restriction endonuclease, releasing a 
secondary amplification cleavage product comprising the lipophilic label 
linked to fewer hydrophilic nucleotides than are present in the signal 
primer. As a result of its increased hydrophobicity and increased 
lipophilicity, the labeled cleavage product becomes soluble in the organic 
phase, separating label generated as a result of target amplification from 
the label of the unreacted signal primer. When very few nucleotides remain 
linked to the label, the double-stranded secondary amplification cleavage 
product may denature into single strands. This is also the case for nicked 
secondary amplification products, as described below. Longer secondary 
amplification cleavage products may be sufficiently stable to remain 
hybridized. Whether the cleavage product is single- or double-stranded is 
not critical to the invention, as either form will be transferrable to the 
organic phase on the basis of increased lipophilicity. Unreacted 
full-length signal primer, which is more hydrophilic due to the greater 
number of hydrophilic nucleotides linked to the lipophilic label, remains 
in the aqueous phase. Therefore, if there is no target amplification, 
little or no label will be detected in the organic phase. As greater 
levels of target amplification occur, more label will be detected in the 
organic phase. The methods of the invention may therefore be used for 
either qualitative detection of amplification (presence or absence of 
target) or for quantitation of amplification (measuring the amount of 
label in the organic phase to determine the initial amount of target). 
When amplification is by SDA, the restriction endonuclease used to reduce 
the number of nucleotides linked to the lipophilic label may be the same 
restriction endonuclease employed in the SDA reaction (e.g., BsoBI or 
HincII). In this case, the restriction endonuclease recognition site in 
the signal primer may be an alternative recognition site for the 
restriction endonuclease which is not protected from double stranded 
cleavage by incorporation of modified dNTPs during amplification. The 
restriction endonuclease recognition site in the amplification primers, 
however, is a recognition site which is nicked by the restriction 
endonuclease when in hemimodified form. For example, the HincII 
recognition site GTCGAC undergoes double stranded cleavage by HincII even 
when hemimodified by incorporation of .alpha. thio-dATP. This recognition 
site is therefore suitable for use in the signal primer. In contrast, the 
HincII recognition site GTTGAC is nicked when hemimodified by 
incorporation of .alpha.thio-dATP and is therefore suitable for use in the 
amplification primers. Similarly, the BsoBI recognition site CTCGAG 
undergoes double stranded cleavage by BsoBI even when hemimodified with 
.alpha.thio-dCTP and is therefore suitable for use in the signal primer. 
The BsoBI recognition site CTCGGG is nicked when hemimodified with 
.alpha.thio-dCTP and is therefore suitable for use in the amplification 
primers. 
A nickable restriction endonuclease recognition site in the signal primer 
may also be used to generate the secondary amplification products, as very 
short labeled nucleotide segments resulting from nicking would not remain 
hybridized and would be released into the organic phase. If some of the 
short, labeled, nicked segments remain hybridized to the signal primer, 
the nick can be placed close to the 5' end of the signal primer to prevent 
initiation of polymerization and displacement by the polymerase. 
Polymerases, in general, require about 8-10 nucleotides 5' to the nick for 
priming. Therefore, using restriction endonucleases such as HincII or 
BsoBI, the target sequence may be amplified and the number of nucleotides 
in the secondary amplification products may be concurrently reduced by 
means of a single restriction endonuclease. Alternatively, the restriction 
endonuclease for reducing the number of nucleotides in the secondary 
amplification product may be different from the restriction endonuclease 
for SDA. For example, an EcoRI recognition site, or any other recognition 
site which remains cleavable upon incorporation of modified dNTPs during 
SDA, may be included in the signal primer. In this type of reaction, both 
the cleaving restriction endonuclease and the nicking restriction 
endonuclease may be present in the amplification reaction to achieve 
concurrent amplification of target and generation of lipophilic cleavage 
products. Routine screening methods testing a restriction endonuclease 
against its hemimodified recognition site may be used to identify those 
recognition sites which are cleaved and those which are nicked. 
It will be apparent from the reactions illustrated in FIGS. 1 and 2 that, 
in addition to SDA, the methods of the invention are easily adapted to 
other primer extension amplification methods (e.g., PCR or 3SR). For 
example, replacing SDA amplification primers with PCR amplification 
primers and a PCR DNA polymerase which lacks 5'.fwdarw.3' exonuclease 
activity (e.g., Sequencing Grade Taq from Promega or exo.sup.- Vent or 
exo.sup.- Deep Vent from New England BioLabs) in the reaction scheme of 
FIG. 2, secondary amplification products are generated which contain a 
cleavable, double-stranded restriction endonuclease recognition site 
contributed by the signal primer. As thermocycling is a feature of 
amplification by PCR, the restriction endonuclease is preferably added at 
low temperature after the final cycle of primer annealing and extension, 
however, a thermophilic restriction endonuclease which remains active 
through the high temperature cycle of the PCR reaction could be present 
during amplification. As in SDA Systems, cleavage of the restriction 
endonuclease recognition site generates a lipophilic secondary 
amplification product. This secondary amplification product can be 
transferred to an organic phase for detection as an indicator of target 
amplification as herein described. 
In a preferred PCR method according to the invention, lipophilic secondary 
amplification products may be generated from a hydrophilic signal primer 
in a target amplification-dependent manner by employing a lipophilic label 
in the PCR methods of P. M. Holland, et al., supra. Referring to the 
Figure at page 462 of the publication, Taq DNA polymerase extends the 
amplification primer and displaces the first few nucleotides of the 
hybridized downstream probe (i.e., the signal primer), cleaving the signal 
primer at the phosphodiester bond joining the displaced region with the 
remaining base-paired portion of the signal primer. This releases a 
labeled secondary amplification cleavage product with significantly fewer 
nucleotides linked to the lipophilic label (usually 1-2). This cleavage 
product is generated in a target amplification-dependent manner (i.e., 
only upon hybridization and extension of amplification primers and signal 
primers on a target sequence), as the partially double-stranded "fork" 
structure is the preferred substrate for cleavage. The lipophilic label 
with the reduced number of linked nucleotides can then be transferred to 
an organic phase for detection as an indicator of target amplification. As 
generation of lipophilic cleavage products in this system does not require 
that the 5' end of the signal primer be double-stranded, the 3'-end of the 
signal primer may be unextendable as described by the authors. 
Alternatively, the 3' end of the signal primer may be extendable without 
interfering with generation, phase transfer and detection of the 
lipophilic cleavage product. However, an extendable signal primer may 
increase background and extending the signal primer unnecessarily reduces 
the efficiency of the polymerase in amplification. The number of 
nucleotides linked to a selected lipophilic label can be varied by varying 
the nucleotide sequence of the signal primer. Higher A+T content in the 5' 
end of the signal primer facilitates generation of larger, less lipophilic 
cleavage products as a result of more efficient displacement by Taq 
polymerase and/or "breathing" of the duplex before cleavage. Conversely, 
increased G+C content generally reduces the size of the cleavage product 
and increases its lipophilicity. Such routine variation of the sequence of 
the signal primer may therefore be used to optimize the length of the 
cleavage product for a selected label and a selected organic phase. 
Alternatively, the number of nucleotides linked to the lipophilic label in 
the cleavage product may be increased by inclusion of a non-hybridizing 
tail in the signal primer between the lipophilic label and the target 
binding sequence of the signal primer. However, as previously stated, more 
lipophilic secondary amplification products having fewer linked 
nucleotides are generally preferred. 
For adaptation of the inventive methods to 3SR, it is only necessary to 
employ a 5'.fwdarw.3' exonuclease deficient reverse transcriptase with 
strand displacing activity in the 3SR reaction, with hybridization of a 
signal primer to the RNA target downstream of the "3' primer" and/or the 
"5' primer" of Guatelli, et al., supra (see Guatelli's FIG. 1, page 1875). 
In a reaction scheme similar to Applicant's FIG. 1, the hybridized signal 
primer containing the restriction endonuclease recognition sequence is 1) 
extended, and 2) displaced by extension of the upstream DNA primer. The 
displaced extension product is then made double stranded by hybridization 
and extension of the other primer. This renders the restriction 
endonuclease recognition site cleavable, and a lipophilic secondary 
amplification product is generated for transfer to the organic phase. Also 
similar to SDA, the signal primer for 3SR does not contain a T7 RNA 
polymerase promoter sequence and therefore cannot function as an 
amplification primer, reducing nonspecific background signal. 
It will be apparent from the foregoing examples that the essential feature 
of the invention is generation of a lipophilic secondary amplification 
product from a hydrophilic signal primer by a target 
amplification-dependent reduction in the number of nucleotides linked to 
the lipophilic label. Any such secondary amplification product, regardless 
of the reaction mechanism by which it is produced during target 
amplification, will be transferrable to an organic phase as herein 
described for detection as an indicator of target amplification. 
Compounds for use as labels on oligonucleotides are well known in the art, 
and include dyes, enzymes, radiolabels, ligands, antigens/haptens and 
antibodies. Any such labels which are lipophilic and detectable upon 
transfer to the organic phase are suitable for use in the invention. Dyes 
are particularly useful due to the ease with which they can be detected, 
and many colorimetric and fluorescent dyes have the necessary lipophilic 
properties. Examples of such dyes are discussed in Molecular Probes 
Handbook of Fluorescent Probes and Research Chemicals, 5th Edition, by 
Richard P. Haugland, Molecular Probes Inc. (1992) and Fluorescent Probes 
in Cellular and Molecular Biology by Jan Slavik, CRC Press (1994). 
Specific lipophilic dyes suitable for use in the invention include, but 
are not limited to, diphenylhexatriene, Nile Red, 
N-phenyl-1-naphthylamine, Prodan, Laurodan, Pyrene, Perylene, rhodamine, 
rhodamine B, tetramethylrhodamine, Texas Red, sulforhodamine, 
1,1'-didodecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate, octadecyl 
rhodamine B and the BODIPY dyes available from Molecular Probes Inc. 
(e.g., BODIPY 558/568, BODIPY D-3921, BODIPY D-3935, BODIPY D-3933, BODIPY 
B-3930, BODIPY B-3932, BODIPY B-2192, BODIPY B-2188, BODIPY B-2223, BODIPY 
B-2226, BODEPY B-2229, and BODIPY B-2185). For a selected lipophilic 
label, the appropriate number of linked nucleotides in the signal primer 
to obtain hydrophilic properties and the appropriate number of linked 
nucleotides in the secondary amplification product to obtain lipophilic 
properties may be routinely determined using simple screening methods such 
as those described in Example 1. That is, by linking a variable number of 
nucleotides to the selected lipophilic label and testing for solubility in 
an organic phase or an aqueous phase, the practitioner can design a 
suitable signal primer for use in the methods of the invention. The label 
associated with the secondary amplification product may then be detected 
after transfer to the organic phase using methods known in the art 
appropriate for the selected label. In the case of colorimetric dyes, 
absorbance of light is typically used for detection, for example using a 
spectrophotometer. Fluorescent dyes may also be detected by absorbance. 
Alternatively, fluorescent dyes may be excited by an appropriate 
wavelength of light and detected by the emission of fluorescence, for 
example in a fluorometer or by fluorescence spectroscopy. 
Similarly, a variety of organic phases may be screened by routine methods 
to determine solubility of a signal primer or secondary amplification 
product incorporating a selected label. That is, although different signal 
primers and secondary amplification products incorporating different 
lipophlic labels may exhibit different solubility characteristics in 
different organic phases, whether or not a selected organic phase is 
useful in the methods of the invention can be determined using simple 
screening assays such as those described in Example 1. Any organic phase 
in which the secondary amplification product is soluble and the signal 
primer is insoluble (or minimally soluble, preferably below the level of 
detection) is suitable for use in the methods of the invention, e.g., 
chloroform, phenol, butanol, methylene chloride and mixtures thereof. The 
proportions of the components in a mixed organic phase are generally not 
critical, and can be optimized as necessary using simple screening assays 
as described above. The lipophilic secondary amplification product is 
typically transferred to the organic phase by mixing the organic phase 
with the aqueous reaction phase to extract it. The aqueous and organic 
phases are then separated, naturally or assisted by centrifugation, and 
any label extracted into the organic phase is detected. 
If solubility of the lipophilic secondary amplification product in the 
organic phase is inadequate to provide the desired sensitivity or if it is 
otherwise desired to enhance solubility, additives may be included in the 
aqueous phase to increase its ionic strength. By increasing the ionic 
strength of the aqueous phase, the solubility of the lipophilic secondary 
amplification product is reduced. Such additives include compounds which 
ionize in aqueous solution. Such compounds include, but are not limited 
to, mineral acids (e.g., phosphoric acid, hydrochloric acid, sulfuric 
acid), organic acids (e.g., acetic acid and other carboxylic acids), 
chloride salts (e.g., sodium, potassium, calcium, or magnesium chlorides), 
carbonate salts (e.g., sodium, potassium, calcium or magnesium carbonate), 
sulfate salts (e.g., sodium, potassium, calcium or magnesium sulfate) and 
phosphate salts (e.g., sodium, potassium, calcium or magnesium phosphate). 
For any selected combination of signal primer, secondary amplification 
product and organic phase, the amount of the selected additive to be 
included in the aqueous phase to enhance transfer of the lipophilic 
secondary amplification product to the organic phase may be determined by 
routine screening assays in which transfer is monitored in the presence of 
a range of aqueous phase ionic strengths. 
EXAMPLE 1 
A series of dye-conjugated oligodeoxynucleotides were screened for their 
solubility in a variety of organic phases. Short oligodeoxynucleotides of 
varying length were synthesized and labeled at the 5'-end with BODIPY 
558/568 (Molecular Probes, Inc. catalog #2218) as recommended by the 
manufacturer: 
__________________________________________________________________________ 
dye-1-mer 
5'-BODIPY-dG 
dye-2-mer 
5'-BODIPY-dGG 
dye-3-mer 
5'-BODIPY-dGGA 
dye-4-mer 
5'-BODIPY-dGGAA 
dye-33-mer 
5'-BODIPY-dGGAATTCATCCGTATGGTGGATAACGTCTTTCA 
(SEQ ID NO: 1) 
__________________________________________________________________________ 
The labeled oligodeoxynucleotides and the free dye were then tested for 
their ability to partition from an aqueous phase into each of several 
organic phases. The oligos or free dye were dissolved in 100 .mu.L of 50 
mM K.sub.2 PO.sub.4, pH 7.5, and an equal volume of organic solvent was 
added. The samples were mixed by vortexing and the two phases were 
separated by centrifugation. The amount of BODIPY-oligodeoxynucleotide (or 
free dye) in each of the two phases was determined by fluorescence 
spectroscopy. The percentage of BODIPY-oligodeoxynucleotide (or free dye) 
transferred from the aqueous phase to the organic phase is shown in Table 
I. 
TABLE I 
______________________________________ 
DYE- PHENOL/ METH- 
OLI- CHLORO- BU- CHLOROFORM YLENE 
GO FORM TANOL (50:50) CHLORIDE 
______________________________________ 
Dye 92% 90% 79% 81% 
Alone 
Dye- 18% 71% 78% 5% 
1-mer 
Dye- 5% 7% 45% 7% 
2-mer 
Dye- 5% 3% 26% 5% 
3-mer 
Dye- 3% 2% 5% 4% 
4-mer 
Dye- 6% 4% 3% 10% 
33-mer 
______________________________________ 
In this experiment, phenol/chloroform was particularly well suited for 
extracting BODIPY-labeled 1-3-mers. Butanol worked well for extraction of 
BODIPY-labeled 1-mer. None of the organic phases successfully extracted 
BODIPY-labeled 33-mer or BODIPY-labeled 4-mer. This screening assay 
demonstrates that for this dye, a 1, 2, or 3-mer secondary amplification 
product will be transferred from the aqueous phase to phenol/chloroform 
(or butanol if the secondary amplification product is a 1-mer). In 
addition, a signal primer which is at least four nucleotides in length is 
sufficiently hydrophilic in this system to remain in the aqueous phase. 
Various aqueous phase additives were tested in the assay to determine their 
ability to enhance phenol/chloroform extraction of the dye-3-mer without 
resulting in extraction of the dye-33-mer. The dye-oligodeoxynucleotides 
or the free dye were dissolved in 100 .mu.L of 50 mM K.sub.2 PO.sub.4, pH 
7.5 and 10 .mu.L of the indicated acid or salt was added. An equal volume 
of phenol/chloroform (50:50) was added, and the phases were mixed by 
vortexing and separated by centrifugation. The amount of 
BODIPY-oligodeoxynucleotide or free dye in each of the two phases was 
determined by fluorescence spectroscopy. The percentage of 
BODIPY-oligodeoxynucleotide or free dye transferred from the aqueous phase 
to the organic phase is shown in Table II. 
TABLE II 
______________________________________ 
DYE- CONCENTRAT- CONCENTRATED 
OLI- ED ACETIC 0.1M PHOSPHORIC 5M 
GO ACID HCl ACID NaCl 
______________________________________ 
Dye 97% 98% 97% 98% 
Alone 
Dye- 96% 97% 96% 85% 
3-mer 
Dye- 2% 11% 5% 1% 
33-mer 
______________________________________ 
As compared to the solvent alone, all four additives substantially 
increased extraction of the dye-3-mer into the organic phase (to 
essentially the level of the dye alone) without any appreciable increase 
in extraction of the dye-33-mer. Addition of such additives should be 
generally applicable, and would be expected to increase extraction of the 
smaller BODLPY-oligonucleotides as well Further, addition of additives to 
increase the ionic strength of the aqueous phase would be expected to 
increase transfer of secondary amplification products to other organic 
phases as well. 
EXAMPLE 2 
Oligodeoxynucleotides were synthesized on an Applied Biosystems Inc. DNA 
synthesizer (Model 380B) and were purified by denaturing gel 
electrophoresis. An oligodeoxynucleotide signal primer was synthesized for 
hybridization to nucleotides 985-1010 of the IS6110 element of M. 
tuberculosis (Thierry, et al. 1990. Nucl. Acids Res. 18, 188). This 
sequence is within the IS6110 sequence to be amplified (nucleotides 
970-1025 of the IS6110 element). The signal primer sequence was as follows 
: 
EQU 5'dGC/TCGAGTTGTCTACATCCGTATGGTGGATAACGTCTTTCA (SEQ ID NO:2) 
The signal primer was 5'-end labeled with BODIPY 558/568 (Molecular Probes, 
Inc.) as recommended by the manufacturer. The following amplification and 
bumper primers were also synthesized: 
S.sub.1 amplification primer (SEQ ID NO:3) 
EQU 5'dCGATTCCGCTCCAGACTTCTCGGGTGTACTGAGATCCCCT 
S.sub.2 amplification primer (SEQ ID NO:4) 
EQU 5'dACCGCATCGAATGCATGTCTCGGGTAAGGCGTACTCGACC 
B.sub.1 bumper primer (SEQ ID NO:5) 
EQU 5'dCGCTGAACCGGAT 
B.sub.2 bumper primer (SEQ ID NO:6) 
EQU 5'dTGGACCCGCCAAC 
BsoBI sites are indicated in bold italics and the cleavage site is 
indicated by the slash in SEQ ID NO:1. The target binding regions are 
underlined. 
Strand Displacement Amplification was performed generally as described by 
Walker, et al. (1992. Nucl. Acids Res., supra). Samples for amplification 
were initially prepared as 35 .mu.L of 50 mM K.sub.2 PO.sub.4 (pH 7.5), 
10.7 mM MgCl.sub.2, 2 mM each dGTP, dATP, TTP and .alpha.thio-dCTP, 0.14 
mg/mL bovine serum albumin, 143 nM primer S.sub.1,714 nM primer S.sub.2, 
57 nM each primers B.sub.1 and B.sub.2, and 29 nM 5'-BODIPY-labeled signal 
primer. Varying amounts of M. tuberculosis target DNA were then added to 
each sample in 5 .mu.L aliquots of 10 mM TRIS-HCl (pH 7.9), 10 mM 
MgCl.sub.2, 50 mM NaCl, 1 mM DTT and 500 ng human placental DNA. These 40 
.mu.L samples were denatured by heating for 2 min. in a boiling water bath 
and equilibrated for 3 minutes at 60.degree. C. for primer annealing. 
BsoBI and a 5'-3' exonuclease deficient form of DNA polymerase from 
Bacillus caldotenax (5'-3' exo.sup.- Bca, PanVera) were diluted together 
to 16 units/.mu.L and 0.4 units/.mu.L, respectively, in 10 mM TRIS-HCI (pH 
7.9), 10 mM MgCl.sub.2, 50 mM NaCl, 1 mM DTT. The enzyme mixture was 
prepared at room temperature immediately before addition of a 10 .mu.L 
aliquot to each of the equilibrated 40 .mu.L samples. The final 50 .mu.L 
reaction mixture contained 35 mM K.sub.2 PO.sub.4 (pH 7.5), 3 mM TRIS-HCL 
(pH 7.9), 15 mM NaCl, 0.3 mM DTT, 105 mM MgCl.sub.2, 1.4 mM each dGTP, 
dATP, TTP and .alpha.thio-dCTP, 0.1 mg/mL bovine serum albumin, 500 ng 
human placental DNA, 100 nM primer S.sub.1, 500 nM primer S.sub.2, 40 nM 
each primer B.sub.1 and B.sub.2, 160 units BsoBI (New England BioLabs), 4 
units of 5'-3' exo.sup.- Bca, 20 nM 5'-BODIPY-labeled signal primer and 
varying amounts of M. tuberculosis DNA. 
After enzyme addition, the SDA reaction was allowed to proceed for 30 min. 
at 60.degree. C. and was terminated by addition of 3 .mu.L of 0.5M EDTA. 
Samples were then diluted with 350 .mu.L of 0.5M NaCl, 10 mM TRIS-HCl (pH 
7.4) and extracted with 400 .mu.L of phenol/chloroform/isoamyl alcohol 
(25:24: 1). The fluorescence intensity of the organic phase was measured 
at 579 nm after excitation at 556 nm. Fluorescence intensity values for 
the organic phase for each initial M. tuberculosis target level are 
presented in Table III. Increased fluorescence intensity above background 
indicates target-specific conversion of the single-stranded signal primer 
to a double-stranded form which was cleaved by BsoBI, resulting in release 
and transfer of a BODIPY-labeled dinucleotide to the organic phase. All M. 
tuberculosis levels tested were detectable over the negative control 
sample which contained no M. tuberculosis target DNA. Increasing 
fluorescence with increasing levels of target demonstrated that the 
inventive methods can be used for quantitation of target in amplification 
reactions. 
TABLE III 
______________________________________ 
Organic Phase 
# M. tuberculosis 
Fluorescence 
Target Genomes Intensity 
______________________________________ 
50000 100842 
5000 91485 
500 86334 
0 75253 
______________________________________ 
EXAMPLE 3 
SDA is performed as described by Walker, et al. (1992. Nucl. Acids Res., 
supra), using the S.sub.1, S.sub.2, B.sub.1 and B.sub.2 primers for 
amplification of the IS6110 sequence disclosed therein. The amplification 
reaction also includes a 5'-BODIPY-labeled signal primer which is 33 
nucleotides in length (5'-dGGAATTCATCCGTATGGTGGATAACGTCTTTCA--SEQ ID 
NO:7). The 26 nucleotides at the 3'-end of the signal primer (the target 
binding sequence) will hybridize to the IS6110 target sequence at 
nucleotide positions 985-1010, between the amplification primers. 5' to 
the target binding sequence is a recognition site for the restriction 
endonuclease EcoRI (bold italics). After SDA, EcoRI is added to the 
amplification reaction and the samples are incubated for at 37.degree. C. 
for a sufficient time to allow cleavage of the secondary amplification 
product. 
During the SDA reaction, the signal primer is extended by the polymerase to 
a length of 49 nucleotides. This 49-mer is displaced by extension of the 
upstream amplification primer. The 3'-end of the 49-mer will hybridize to 
the 3' -end of the other amplification primer, forming a double-stranded 
70-mer after extension by polymerase. The single-stranded EcoRI 
recognition site at the 5'-end of the signal primer will become cleavable 
by EcoRI upon formation of the double-stranded 70-mer. EcoRI cleavage of 
the double-stranded 70-mer will produce a cleavage product which is a 
5'-BODIPY-labeled dinucleotide. This dinucleotide is detectable as a 
secondary amplification product by virtue of the linked dye. 
The cleaved secondary amplification product (the BODIPY-labeled 
dinucleotide) is detected by mixing the aqueous reaction with 
phenol/chloroform and separating the aqueous and organic phases, e.g., by 
centrifugation. Fluorescence in the organic phase is then detected, e.g., 
by fluorescence spectroscopy. An increase in fluorescence transferred to 
the organic phase after amplification (as compared to an unamplified 
control) will indicate that the IS6110 target sequence is present and has 
been amplified. If no increase in fluorescence is detected in the organic 
phase, the target sequence is not present or is present but has not been 
amplified. 
EXAMPLE 4 
The IS6110 target sequence is amplified by PCR with generation of a 
secondary amplification product as described by P. M. Holland, et al., 
supra. The amplification primer pair consists of the target binding 
regions of SEQ ID NO:3 and SEQ ID NO:4, or the amplification primer pair 
may consist of the target binding regions of the S.sub.1 and S.sub.2 
primers of Walker, et al. (1992. Nucl. Acids Res., supra). The signal 
primer included in the amplification reaction is labeled at the 5'-end 
with a lipophilic dye and has a sequence based on the target binding 
region of SEQ ID NO:2 (which is identical to the target binding region of 
SEQ ID NO:7). The sequence of the signal primer may include the entire 
target binding region, but is preferably a segment of the target binding 
region which is GC-rich at the 5'-end. As the 5'-end of the target binding 
region of SEQ ID NO:2 and SEQ ID NO:7 begins with the sequence ATCCG, it 
may be preferable to eliminate the 5'-AT and begin the signal primer 
sequence with 5'-CCG. Designing a signal primer with a GC-rich region at 
the 5'-end promotes the production of smaller polymerase cleavage products 
(mono- and dinucleotides), thereby ensuring that the cleavage product will 
be sufficiently lipophilic for transfer to the organic phase. 
Alternatively, the 5' end of the signal primer may comprise a very short 
nucleotide "tail" which does not hybridize to the target sequence 
(preferably no more than about 1-3 nucleotides in length) to that the 
polymerase encounters the preferred "fork" structure without the need to 
displace hybridized nucleotides in the signal primer. 
The IS6110 target sequence is amplified by PCR in the presence of the 
amplification primers and the signal primer, essentially as described by 
R. K. Saiki, et al. (1985. Science 230, 1350-1354) and K. B. Mullis, et 
al. (1987. Methods Enzymol. 155, 335-350) utilizing the 5'.fwdarw.3' 
exonuclease activity of Taq DNA polymerase to generate 
target-amplification specific signal primer cleavage products as described 
by Holland, et al., supra. After stopping the amplification reaction, an 
organic solvent is added to the aqueous reaction phase and mixed. The 
phases are separated (e.g., by centrifugation) and the organic phase is 
assayed for presence of the dye using methods appropriate for detection of 
the selected dye. If an increase in the amount of dye transferred to the 
organic phase as compared to an unamplified control reaction is detected, 
the target sequence is present and has been amplified. If no increase is 
detected, the target sequence is not present or is present but has not 
been amplified. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 7 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GGAATTCATCCGTATGGTGGATAACGTCTTTCA33 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GCTCGAGTTGTCTACATCCGTATGGTGGATAACGTCTTTCA41 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CGATTCCGCTCCAGACTTCTCGGGTGTACTGAGATCCCCT40 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ACCGCATCGAATGCATGTCTCGGGTAAGGCGTACTCGACC40 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CGCTGAACCGGAT13 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TGGACCCGCCAAC13 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GGAATTCATCCGTATGGTGGATAACGTCTTTCA33 
__________________________________________________________________________