Method for detection of nucleic acid targets by amplification and fluorescence polarization

Fluorescence polarization methods for detection of nucleic acid amplification at thermophilic temperatures employ a fluorescently labeled oligonucleotide signal primer which is converted from single- to double-stranded form in a target amplification-dependent manner. This conformational change is accompanied by an increase in fluorescence polarization values. The decrease in FP typically observed for the duplex at elevated temperatures is overcome by double-stranded DNA binding proteins which are believed to stabilize the double-stranded structure by reducing the single-strandedness normally associated with higher temperatures. The inventive methods provide a closed, homogeneous system for amplification and detection of amplification in real-time or at an endpoint.

FIELD OF THE INVENTION 
The present invention relates to methods for detecting amplification of 
nucleic acid target sequences and in particular to detection of 
amplification by fluorescence polarization. 
BACKGROUND OF THE INVENTION 
Fluoresence Polarization (FP) is a measure of the time-average rotational 
motion of fluorescent molecules. It has been known since the 1920's and 
has been used in both research and clinical applications for sensitive 
determination of molecular volume and microviscosity. The FP technique 
relies upon changes in the rotational properties of molecules in solution. 
That is, molecules in solution tend to "tumble" about their various axes. 
Larger molecules (e.g., those with greater volume or molecular weight) 
tumble more slowly and along fewer axes than smaller molecules. There is 
therefore less movement between excitation and emission, causing the 
emitted light to exhibit a relatively higher degree of polarization. 
Conversely, fluorescence emissions from smaller fluorescent molecules, 
which exhibit more tumbling between excitation and emission, are more 
multiplanar (less polarized). When a smaller fluorescent molecule takes a 
larger or more rigid conformation its tumbling decreases and the emitted 
fluorescence becomes relatively more polarized. This change in the degree 
of polarization of emitted fluorescence can be measured and used as an 
indicator of increased size and/or rigidity of the fluorescent molecule. 
In fluorescence polarization techniques, the fluorescent molecule is first 
excited by polarized light. The polarization of the emission is measured 
by measuring the relative intensities of emission (i) parallel to the 
plane of polarized excitation light and (ii) perpendicular to the plane of 
polarized excitation light. A change in the rate of tumbling due to a 
change in size and/or rigidity is accompanied by a change in the 
relationship between the plane of excitation light and the plane of 
emitted fluorescence, i.e., a change in fluorescence polarization. Such 
changes can occur, for example, when a single stranded oligonucleotide 
probe becomes double stranded or when a nucleic acid binding protein binds 
to an oligonucleotide. Fluorescence anisotropy is closely related to FP. 
This technique also measures changes in the tumbling rates of molecules 
but is calculated using a different equation. It is to be understood that 
polarization and anisotropy are interchangeable techniques for use in the 
present invention. The term fluorescence polarization is generally used 
herein but should be understood to include fluorescence anisotropy 
methods. In steady state measurements of polarization and anisotropy, 
these values are calculated according to the following equations: 
##EQU1## 
where Ipa is the intensity of fluorescence emission parallel to the plane 
of polarized excitation light and Ipe is the intensity of fluorescence 
emission perpendicular to the plane of polarized excitation light. 
As FP is homogenous, this technique is ideal for studying molecular 
interactions in solution without interference by physical manipulation. 
Fluorescence polarization is therefore a convenient method for monitoring 
conversion of single-stranded fluorescently labelled DNA to 
double-stranded form by hybridization (Murakami, et al. 1991. Nucl. Acids 
Res. 19, 4097-4102). The ability of FP to differentiate between single and 
double-stranded nucleic acid conformations without physical separation of 
the two forms has made this technology an attractive alternative for 
monitoring probe hybridization in diagnostic formats. European Patent 
Publication No. 0 382 433 describes fluorescence polarization detection of 
amplified target sequences by hybridization of a fluorescent probe to the 
amplicons or by incorporation of a fluorescent label into the 
amplification products by target-specific extension of a 
fluorescently-labeled amplification primer. PCT Patent Publication No. WO 
92/18650 describes similar methods for detecting amplified RNA or DNA 
target sequences by the increase in fluorescence polarization associated 
with hybridization of a fluorescent probe. 
Fluorescence polarization may be monitored as either transient state FP or 
steady state FP. In transient state FP, the excitation light source is 
flashed on the sample and polarization of the emitted light is monitored 
by turning on the photomultiplier tube after the excitation light source 
is turned off. This reduces interference from light scatter, as 
fluorescence lasts longer than light scatter, but some fluorescence 
intensity is lost. In steady state FP, excitation light and emission 
monitoring are continuous (i.e., the excitation source and photomultiplier 
tube are on continuously). This results in measurement of an average 
tumbling time over the monitoring period and includes the effects of 
scattered light. 
In vitro and in situ nucleic acid amplification techniques have provided 
extremely sensitive tools for detection and analysis of small amounts of 
nucleic acids. 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. 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, 
and U.S. Pat. No. 5,455,166), self-sustained sequence replication (3SR; J. 
C. Guatelli, et al. 1990. Proc. Natl. Acad. Sci USA 87, 1874-1878), 
Nucleic Acid Sequence Based Amplification (U.S. Pat. No. No. 5,409,818), 
restriction amplification (U.S. Pat. No. 5,102,784) and the Q.beta. 
replicase system (P. M. Lizardi, et al. 1988. BioTechnology 6, 1197-1202) 
are isothermal reactions. Isothermal amplifications are conducted at 
essentially constant temperature, in contrast to the cycling between high 
and low temperatures characteristic of amplification reactions such as the 
PCR. 
Strand Displacement Amplification (SDA) utilizes nicking of a hemimodified 
restriction endonuclease recognition site by a restriction enzyme and 
displacement of a downstream DNA strand by a polymerase to amplify a 
target nucleic acid (U.S. Pat. No. 5,270,184, Walker, et al. 1992. Proc. 
Natl. Acad Sci. USA 89, 392-396; Walker, et al. 1992. Nucl. Acids Res. 20, 
1691-1696). The SDA reaction originally reported in the publications cited 
above ("conventional SDA") is typically conducted at a temperature between 
about 35.degree. C. and 45.degree. C., and is capable of 10.sup.8 -fold 
amplification of a target sequence in about 2 hours. Recently, SDA has 
been adapted for higher reaction temperatures (about 45.degree.-65.degree. 
C.--"thermophilic SDA" or "tSDA"). tSDA is capable of producing 10.sup.9 
-10.sup.10 fold amplification in about 15-30 min. at about 
50.degree.-60.degree. C. In addition to increased reaction speed, there is 
a significant reduction in non-specific background amplification in tSDA 
as compared to conventional SDA. 
Typically, amplified target sequences are detected by post-amplification 
hybridization of probes. This requires separation of free and hybridized 
probe before the signal can be measured. However, monitoring changes in FP 
allows differentiation of free and hybridized probe without physical 
separation and therefore enables amplification and detection of 
amplification in a homogeneous, closed system. A closed, homogeneous assay 
reduces operating steps and procedural complexity, as well as providing 
improved control of the dispersal of amplification products in the 
laboratory, thereby reducing the potential for false positives due to 
accidental contamination of samples with target DNA. FP detection has been 
applied to detection of amplification in methods in which double-stranded 
secondary amplification products are generated during the amplification 
reaction from a single-stranded signal primer comprising a fluorescent 
label. Generation of secondary amplification products during target 
amplification is described and illustrated in published European Patent 
Application Nos. 0 678 582 and 0 678 581. In the process, a 
single-stranded oligonucleotide signal primer comprising a detectable 
label is converted to double-stranded form in a target 
amplification-dependent manner. Hybridization, extension and displacement 
of the signal primer occurs concurrently with the amplification reaction. 
When the label is fluorescent, conversion of the signal primer to 
double-stranded form may be detected as a change in FP. Conversion of the 
signal primer to double-stranded form results in an increase in FP of 
approximately 20 mP using fluorescein or La Jolla Blue as the fluorescent 
label. This change in FP can be enhanced (e.g., to about 133-185 mP) by 
binding a double-stranded DNA binding protein to its specific binding 
sequence present in the secondary amplification product. Enhancement is 
therefore amplification-specific because protein binding can only occur 
once the binding sequence in the secondary amplification product becomes 
double-stranded as a result of target amplification. 
The speed and specificity of target amplification is increased at higher 
reaction temperatures (typically about 45.degree.-75.degree. C.). It is 
therefore desirable to combine the advantages of FP for detecting 
amplification with elevated amplification temperatures. However, increased 
reaction temperatures were predicted to be incompatible with measurement 
of fluorescence polarization. Many fluorescent labels are not stable at 
higher temperatures. In addition, higher temperatures promote "breathing" 
of the duplex and "fraying" of the ends, leading to increased 
single-strandedness. Increased single-strandedness near the fluorescent 
label, particularly at the ends of the duplex, could result in significant 
decreases in FP for the double-stranded form, potentially eliminating any 
amplification-associated increase in FP under these reaction conditions. 
These concerns were supported by preliminary experiments evaluating FP at 
55.degree. C. At this temperature (which is a typical reaction temperature 
for tSDA), there was no difference in FP between the single-stranded and 
double-stranded forms of oligonucleotides. Further, FP is sensitive to 
sample viscosity, which is altered at higher temperatures. The effects of 
altered sample viscosity on the ability to use changes in FP for detection 
of amplification at increased reaction temperatures were therefore 
uncertain. 
The following terms are defined herein as follows: 
An amplification primer is a primer for amplification of a target sequence 
by hybridization and extension of the primer. For SDA, the 3' end of the 
amplification primer is a target binding sequence which hybridizes at the 
3' end of the target sequence. The amplification primer further comprises 
a recognition site for a restriction endonuclease 5' to the target binding 
sequence, generally near its 5' end. The restriction endonuclease 
recognition site is a nucleotide sequence recognized by a restriction 
endonuclease which will nick a double stranded recognition site for the 
restriction endonouclease when the recognition site is hemimodified, as 
described by Walker, et al. (1992a), supra. For amplification methods 
which do not require specialized sequences at the ends of the target, the 
amplification primer generally consists essentially of only the target 
binding sequence. Amplification methods which require specialized, 
non-target binding sequences other than the nickable restriction 
endonuclease recognition site of SDA (e.g., 3SR) may employ amplification 
primers comprising a target binding sequence and the sequence or structure 
required by the selected amplification method. 
The terms target or target sequence refer to nucleic acid sequences (DNA 
and/or RNA) to be amplified. These include the original nucleic acid 
sequence to be amplified and its complementary second strand as well as 
copies of either strand of the original target sequence produced during 
amplification. These copies also serve as amplifiable target sequences by 
virtue of the fact that they contain copies of the original target 
sequences to which the amplification primers hybridize. 
Amplification products or amplicons are oligo or polynucleotides which 
comprise copies of the target sequence produced during amplification of 
the target sequence. 
A signal primer hybridizes to the target sequence downstream of an 
amplification primer and is extended by polymerase in a manner similar to 
extension of the amplification primer. A signal primer may also be 
referred to as a detector probe. Extension of the amplification primer 
displaces the downstream, extended signal primer from the target sequence. 
The opposite amplification primer then hybridizes to the extended, 
displaced signal primer and is extended by polymerase, resulting in 
incorporation of the signal primer into a longer duplex indicative of 
target amplification (the secondary amplification product). The secondary 
amplification products comprise an internal segment of the amplification 
product and a detectable label which is associated with the signal primer. 
SUMMARY OF THE INVENTION 
The present invention provides methods using FP and a signal primer 
comprising a fluorescent label for detection of nucleic acid amplification 
at elevated temperatures (thermophilic amplification, e.g., at about 
45.degree.-75.degree. C.). Amplification is detected as an increase in FP 
associated with target amplification-dependent generation of 
double-stranded, fluorescent secondary amplification products from the 
single-stranded signal primer. Although preliminary experiments indicated 
that the increased single-strandedness of the secondary amplification 
products at higher temperatures would severely reduce or eliminate the 
increase in FP associated with target amplification, it was unexpectedly 
found that under the conditions of thermophilic target amplification the 
increase in FP is maintained. This phenomenon appears to be due to binding 
of the double-stranded DNA binding proteins in the amplification reaction 
to the double-stranded secondary amplification product, resulting in 
stabilization of the double-stranded form. Further, it has been found that 
when the target is amplified in a thermophilic amplification reaction, 
sequence non-specific double-stranded DNA binding proteins such as the 
amplification polymerase can enhance the change in fluorescence 
polarization associated with target amplification-dependent generation of 
secondary amplification products, regardless of whether fluorescence 
polarization is measured at low temperatures (less than about 45.degree. 
C.) or at thermophilic temperatures (about 45.degree.-75.degree. C.).

DETAILED DESCRIPTION OF THE INVENTION 
Recently developed methods for detection of amplification employ at least 
one signal primer. The signal primer is included in the amplification 
reaction to facilitate detection or monitoring of target amplification. 
During target amplification the signal primer is extended and rendered 
double-stranded as a result of target amplification to produce a secondary 
amplification product (EP 0 678 582 and EP 0 678 581). Conversion of the 
single-stranded signal primer to double-stranded form in the secondary 
amplification product is an indication of target amplification, as 
secondary amplification products are not produced in the absence of target 
amplification. Single- to double-stranded conversion of the signal primer 
may be monitored by measuring fluorescence polarization or fluorescence 
anisotropy when the label of the signal primer is fluorescent. That is, 
the decrease in the local mobility of the fluorophore resulting from the 
change in probe conformation (primarily strandedness) results in a 
detectable change in correlation time (tumbling time) for the fluorescent 
label. The accompanying changes in FP values may be monitored on a 
transient-state fluorometer (e.g., from Diatron) or a steady state 
fluorometer (e.g., Jolley Instruments) appropriate for detection of the 
selected fluorescent label. Fluorescence polarization measurements may be 
taken post-amplification (endpoint measurement) or concurrently with the 
amplification reaction (real-time measurement). Real-time monitoring of 
fluorescence provides significant advantages in the assay. That is, it 
provides an essentially immediate result, it is quantitative, it improves 
sensitivity (analysis of a change in slope is more accurate than a single 
endpoint), and the sample acts as its own internal standard. This last 
advantage is particularly important for analysis of clinical specimens, as 
sample viscosity may significantly affect endpoint readings. 
As the hybridized and unhybridized (i.e., double- and single-stranded) 
signal primers are not separated prior to measurement, FP-based detection 
of target amplification requires appreciable conversion of the 
single-stranded fluorescent signal primer to double-stranded form. 
Therefore, lower signal primer concentrations facilitate high sensitivity 
(ie., detection of amplification of initially low concentrations of target 
sequence) because they result in a higher percentage of converted signal 
primer for a given level of target amplification. However, low signal 
primer concentrations present a kinetic challenge for the amplification 
reaction. The fluorescent signal primer must hybridize to the target 
strand before hybridization and extension of the upstream amplification 
primer. It is therefore generally advantageous to adjust the relative 
primer concentrations such that the signal primer is at a lower 
concentration than the amplification primer which displaces it, and the 
signal primer displacing amplification primer is at a lower concentration 
than the amplification primer which is not displacing a signal primer. 
The processes illustrated in EP 0 678 582 and EP 0 678 581 occur 
concurrently with the amplification reaction and do not interfere with it. 
In SDA, any mispriming by the signal primer and an amplification primer 
generates an extension product which cannot be exponentially amplified due 
to the presence of only one nickable restriction endonuclease recognition 
site (i.e., the fluorescent signal primer does not contain a nickable 
restriction endonuclease recognition site). Non-specific increases in FP 
due to signal primer mispriming are therefore negligible, as SDA requires 
two primers (each containing a nickable restriction endonuclease 
recognition site) to support exponential amplification. This is in 
contrast to the Polymerase Chain Reaction, in which any extendible 
oligonucleotide which hybridizes can serve as an amplification primer, 
allowing misprimed products to be exponentially amplified. Background from 
mispriming by the signal primer is further reduced when the signal primer 
is present at low concentrations (e.g., 50 pM-20 nM). Reduced signal 
primer concentration and their inability to function as amplification 
primers probably make a relatively large contribution to the reduction of 
non-specific background due to signal primer mispriming in conventional 
SDA. However, because the increased reaction temperatures of tSDA 
typically result in significantly lower levels of mispriming than in 
conventional SDA, the effect of signal primer concentration and structure 
are difficult to assess in this system. 
At typical temperatures for isothermal nucleic acid amplification (e.g., 
35.degree.-42.degree. C.) the conversion of 5' fluorescein-labeled signal 
primer from single-stranded to double-stranded form produces an easily 
detectable increase in FP of about 20 mP. This increase can be enhanced by 
addition of a sequence-specific double-stranded DNA binding protein such 
as a restriction endonuclease, repressor protein, receptor binding 
protein, etc. By incorporating the appropriate recognition site for the 
double-stranded DNA binding protein into the signal primer, the 
recognition site becomes double-stranded as a result of target 
amplification, allowing amplification-specific binding of the protein and 
enhancement of the change in FP. At lower temperatures specific protein 
binding sequences are necessary to ensure protein binding exclusively to 
secondary amplification products. This is believed to be due to the 
relatively high levels of non-specific priming at lower temperatures. 
Without specific recognition sequences, it is believed that the 
double-stranded DNA binding protein binds to non-specific background 
amplification products in sufficient amounts to eliminate any 
amplification-specific enhancement of the change in FP. 
Preliminary experiments suggested that the increase in FP associated with 
generation of secondary amplification products would decrease with 
increasing temperature for end-labeled signal primers, and that it would 
be substantially eliminated at the reaction temperatures typical of 
thermophilic amplification reactions such as PCR and tSDA. In nucleic acid 
hybridization studies, the change in FP (.DELTA.mP) was substantially 
unaffected at temperatures below about 45.degree. C. However, .DELTA.mP 
began to decrease at about 45.degree. C. and was essentially absent as 
hybridization temperatures approached about 60.degree. C. However, it was 
unexpectedly found that the change in FP could be maintained when 
generation of secondary amplification products was monitored in 
thermophilic amplification reactions in the temperature range at which FP 
values were negatively affected in hybridization studies, e.g., about 
45.degree.-75.degree. C. As the polymerases used to amplify nucleic acid 
targets are double-stranded nucleic acid binding proteins, Applicants 
believe that this phenomenon is due to sequence-nonspecific binding of the 
amplification polymerase to the secondary amplification products. Binding 
of the polymerase may contribute to stabilization of the duplex and 
reduction or elimination of the increased single-strandedness associated 
with higher hybridization temperatures. This results in a 
target-amplification specific increase in FP which would not be predicted 
from simple hybridization experiments conducted at similar temperatures. 
In addition, it was unexpectedly found that amplification at higher 
temperatures permits the use of sequence non-specific double-stranded DNA 
binding proteins for enhancement of amplification-associated changes in 
FP. Conventional SDA, in contrast to tSDA, did not exhibit any enhancement 
of FP in the presence of the sequence-nonspecific DNA binding proteins in 
the amplification reaction. The different results in these two 
amplification systems may be due to the increased amount of nonspecific 
background amplification product produced by conventional SDA. These 
double-stranded background amplicons could prevent detection of 
enhancement of the change in FP by binding substantial amounts of any 
sequence non-specific double-stranded DNA binding protein which may be 
present. The substantial absence of background amplification in tSDA may 
therefore allow detection of enhanced changes in FP by ensuring that the 
sequence non-specific double-stranded DNA binding protein is primarily 
bound by secondary amplification products. FP detection of amplification 
in thermophilic amplification systems is therefore significantly 
simplified, as there is no need to engineer specific binding sequences 
into the signal primer and the requirement for an additional reaction 
component (a separate double-stranded DNA binding protein) is eliminated. 
That is, the enzymes already present for target amplification (e.g., 
polymerase) stabilize the double-stranded secondary amplification product 
to maintain the change in FP at higher temperatures and may also serve to 
enhance the increase in FP which indicates target amplification. That is, 
under the reaction conditions of thermophilic amplification, the FP 
increase is maintained at levels at least comparable to those observed at 
about 37.degree. C., except for minor changes in magnitude due changes in 
sample viscosity. 
The Strand Displacement Amplification target generation and amplification 
reaction schemes are illustrated in Walker, et al., supra, U.S. Pat. No. 
5,455,166 and U.S. Pat. No. 5,270,184. These general reaction schemes are 
the same for both conventional SDA and tSDA. However, tSDA employs 
thermostable restriction endonucleases and polymerases and is conducted at 
higher temperatures. SDA requires a polymerase which lacks 5'-3' 
exonuclease activity, initiates polymerization at a single stranded nick 
in double stranded nucleic acids, and displaces the strand downstream of 
the nick while generating a new complementary strand using the un-nicked 
strand as a template. Displacement activity is essential to the 
amplification reaction, as it makes the target available for synthesis of 
additional copies and generates the single-stranded extension product to 
which a second amplification primer may hybridize in exponential 
amplification reactions. Examples of polymerases which are thermostable 
and have the other characteristics required for use in tSDA are exo.sup.- 
Vent (New England BioLabs), exo.sup.- Deep Vent (New England BioLabs), Bst 
(BioRad), exo.sup.- Pfu (Stratagene), Bca (Panvera) and Sequencing Grade 
Taq (Promega). Others may be identified using routine screening assays. 
The polymerases Tth (Boehringer), Tfl (Epicentre), REPLINASE (DuPont) and 
REPLITHERM (Epicentre) strand displace from a nick, but also have 5'-3' 
exonuclease activity. These polymerases are useful in tSDA after removal 
of the exonuclease activity, e.g., by genetic engineering. Most of the 
thermophilic polymerasees identified so far have optimal activity at 
65.degree.-76.degree. C. However, as the thermostability of thermophilic 
restriction endonucleases is generally limited to less than about 
65.degree. C., thermophilic polymerases with optimal activity at lower 
temperatures (e.g., Bst and Bca) are more compatible with thermophilic 
restriction endonucleases for use in tSDA. 
Nicking by the restriction endonuclease perpetuates the SDA reaction, 
allowing subsequent rounds of target amplification to initiate. Because 
restriction enzymes generally produce double strand breaks, cleavage of 
one of the two strands in the duplex of the cleavage site must be 
selectively inhibited. This is usually accomplished by introducing 
nucleotide analogs (e.g., deoxynucleoside phosphorothioates) into one 
strand of the DNA during synthesis so that one of the two strands is no 
longer susceptible to cleavage. Examples of restriction endonucleases 
suitable for use in tSDA include BsrI, BstNI, BsmAI, BslI and BsoBI (New 
England BioLabs), and BstOI (Promega). Others may be identified in routine 
screening assays to identify nicking activity at the temperatures of tSDA. 
The present disclosure uses tSDA as an example of thermophilic 
amplification, however, the invention may also be applied to any 
amplification method in which a strand-displacing polymerase is used or in 
which a strand-displacing polymerase can be substituted for a polymerase 
which has 5'-3' exonuclease activity to displace a downstream signal 
primer. The inventive methods may therefore be used in isothermal 
amplification reactions other than SDA, e.g., Self-Sustained Sequence 
Replication (3SR), as the detection method is independent of whether the 
target sequence is RNA or DNA. In 3 SR, target-dependent generation of 
double-stranded signal primer occurs generally as it does for SDA. The T7 
RNA polymerase used in 3SR lacks 5'-3' exonuclease activity and the 
degradative activity of reverse transcriptase is an RNAse H activity which 
is active only on RNA hybridized to DNA. Therefore, in the 3SR 
amplification scheme of Guatelli, et al. (1990. 87, 1874-1878), the signal 
primer may hybridize to the RNA target sequence and be displaced by 
extension of the 3' amplification primer ("A" in FIG. 1 of Guatelli, et 
al.). Alternatively, the signal primer may hybridize to the cDNA target 
sequence generated by reverse transcription in the 3 SR reaction. In 
either case, the extended signal primer is displaced by the polymerase 
when the upstream 3' ("A") or 5' ("B") amplification primer is extended. 
The opposite amplification primer then binds to the signal primer 
extension product and is extended, converting the labeled signal primer to 
double-stranded form. The Transcription Mediated Amplification (TMA) and 
Nucleic Acid Sequence Based Amplification (NASBA) reactions are 
essentially the same as 3SR and would perform similarly to produce 
double-stranded target amplification-specific secondary amplification 
products with addition of a signal primer. Although 3 SR and related 
amplification methods are currently conducted at temperatures below the 
thermophilic temperature range (i.e., less than about 
45.degree.-75.degree. C.), substitution of thermostable enzymes as 
necessary should allow fluorescence polarization detection of 
amplification under thermophilic conditions according to the present 
invention, as all of these amplification reactions include a sequence 
non-specific double-stranded DNA binding protein which would stabilize 
duplexes and maintain FP changes at the higher temperatures. 
The inventive methods may also be applied to detecting amplification in the 
Polymerase Chain Reaction (PCR), although fluorescence polarization 
measurements must be taken during the lower temperature periods of the 
amplification cycle for "real time" monitoring of amplification. Primer 
hybridization and extension in PCR are typically conducted at reduced but 
still thermophilic temperatures (about 60.degree.-75.degree. C.). Using a 
5'-3' exonuclease deficient polymerase (e.g., exo.sup.- Vent, exo.sup.- 
Pfu, or the Stoffel fragment of Taq), extension of a PCR amplification 
primer hybridized to the target sequence displaces the extended downstream 
signal primer. The opposite PCR amplification primer hybridizes to the 
extension product of the signal primer and is extended, resulting in 
conversion of the single-stranded signal primer to double-stranded form. 
The double-stranded signal primer is amplifiable by hybridization and 
extension of one amplification primer and one signal primer in subsequent 
cycles, providing an additional source of double-stranded signal primer. 
The increase in fluorescence polarization or fluorescence anisotropy may 
then be detected after conclusion of the PCR under conditions in which 
amplification products remain double-stranded (below about 45.degree. C.). 
Alternatively, secondary amplification products may be detected during PCR 
at the lower temperature points of the cycling protocol 
(60.degree.-75.degree. C.) with the amplification polymerase serving to 
stabilize the double-stranded structure of secondary amplification 
products and maintain a detectable change in FP. 
As an alternative to using a signal primer, the amplification primers of 
any of the foregoing amplification methods may be fluorescently labeled. 
Double-stranded fluorescently-labeled amplification products are generated 
from the single-stranded amplification primers with an associated change 
in FP. Because background will be higher in this embodiment, sensitivity 
may be reduced as compared to use of a signal primer. 
Any fluorescent molecule known in the art for labeling nucleic acids may be 
used in the methods of the invention, for example, fluorescein and 
fluorescein derivatives such as 5-(4,6-dichlorotriazin-2-yl) amino 
fluorescein (5-DTAF); eosin; rhodamines such as Texas Red and 
tetramethylrhodamine; cyanine dyes such as thiazole orange, oxazole yellow 
and related dyes described in U.S. Pat. Nos. 4,957,870 and 4,888,867; 
pyrene; porphyrin dyes such as La Jolla Blue. The fluorescent label should 
be selected such that its fluorescent lifetime is comparable in magnitude 
to the correlation time being measured, taking into account that 
temperature, viscosity, and the size of the oligonucleotide to which the 
fluorescent dye is conjugated all affect tumbling time. For example, 
fluorescein (lifetime approximately 4 nanosec.) and LaJolla Blue (lifetime 
approximately 2 nanosec.) are both useful for correlation times of about 
0.1-100 nanosec. If a nucleic acid binding protein is used in conjunction 
with the fluorescent label, the correlation time is generally increased. 
For example, correlation time for a free fluorescein label is about 0.2 
nanosec. The correlation time increases to about 0.4 nanosec. when the 
fluorescein label is conjugated to a single stranded oligonucleotide and 
increases further to about 2 nanosec. when conjugated to a double-stranded 
oligonucleotide. When FP is enhanced by binding the fluorescein-labeled 
double-stranded oligonucleotide with a double-stranded DNA binding protein 
such as EcoRi, the correlation time increases again to about 20 nanosec. 
La Jolla Blue (Devlin, et al. 1993. Clin. Chem. 39, 1939-1943) is 
particularly useful for labeling the fluorescent signal primer when 
biological samples are to be amplified, as this dye absorbs and emits 
light in the near-infra red spectrum, a region of relatively low 
background fluorescence with clinical specimens (peak maxima at about 685 
nm and 705 nm, respectively). It has also been found that 5-DTAF is 
superior to fluorescein for FP analysis when used as a label for nucleic 
acids. This label provides a significantly increased dynamic range as 
compared to fluorescein and improves the sensitivity of the FP assay. 
The fluorescent label is covalently linked or conjugated to the signal 
primer so as not to interfere with either emission of fluorescence from 
the label or hybridization of the probe to the target sequence. As FP 
changes occur when the label is near or involved in a conformational 
change, the linkage should be in proximity to the site where the 
conformational change is expected. This is generally at either the 5' end 
of the signal primer or at an internal site. In general, the label is not 
linked to the 3' end of the signal primer, as the 3' end must be available 
for extension by polymerase. A more rigid linkage or "tether", such as one 
containing double bonds, slows the tumbling time of the fluorescent label 
and allows measurement of longer correlation times. The fluorescent label 
is covalently coupled to the signal primer via a linker or "tether" 
suitable for use in conjugating labels to oligonucleotides, e.g., 
amino-ethyl, amino-hexyl and amino-propyl linking arms (Applied 
Biosystems, Clontech, Glen Research, Devlin, et al., supra.). Other amino 
linkers are described in WO 92/18650. The label may also be conjugated to 
the oligonucleotide at C5 of pyrimidines or C8 of purines, as generally 
described by Goodchild, 1990. Bioconj. Chem. 1, 165. Fluorescein may be 
linked internally by synthesis of an oligonucleotide containing a 
phosphorothioate, and subsequent reaction with iodoacetamidofluorescein. 
Methods for linking 5-DTAF to oligonucleotides typically involve reaction 
of an amino-modified oligonucleotide with 5-DTAF in a NaHCO.sub.3 
/Na.sub.2 CO.sub.3 buffer. The labeled oligonucleotide is purified from 
unreacted excess dye by column chromatography and unlabeled 
oligonucleotide is removed to produce the final product. 
It should be noted that when a change in FP is used for detection of 
amplification in real-time (i.e., concurrently with conversion of the 
signal primer to double-stranded form during amplification rather than 
after completion of the reaction), it is not necessary to "zero" the 
sample to compensate for background fluorescence as it is for any endpoint 
measurement. This is because in FP detection a change in polarization or 
the rate of change in polarization (not the absolute magnitude of the 
change) indicates a positive result. Lower concentrations of fluorescently 
labelled signal primer improve detection sensitivity by ensuring that a 
greater percentage of single-stranded signal primer is converted to 
double-stranded form for a given concentration of amplified product. 
However, low signal primer concentrations may result in saturation of the 
signal primer over a broad range of amplified product levels when endpoint 
measurements are taken. End-point measurements of FP, taken after 
completion of the amplification reaction, may therefore not be strictly 
quantitative with regard to the initial target levels. Monitoring FP in 
real-time overcomes the problem of signal primer saturation because 
samples containing higher target levels exhibit more rapid increases in FP 
values than those containing less target. Of course, the correlation 
between the rate of FP increase and initial target levels is valid only 
when comparing samples in which the rate of amplification is essentially 
identical. For clinical specimens, each of which contains varying levels 
of amplification inhibitors, the assay may not be strictly quantitative. 
For example, it may be difficult to differentiate a sample which contains 
a high amount of initial target and undergoes inefficient amplification 
from a sample which contains a low amount of initial target but undergoes 
amplification at a high rate. Nevertheless, real-time monitoring of FP 
values during amplification provides at least a semi-quantitative estimate 
of initial target levels. Quantitation may be improved by including an 
additional target sequence at a known initial concentration as an internal 
positive control (Walker, et al. 1994. Nucl. Acids Res. 22, 2670-2677), or 
running a sample containing the positive control in parallel. The internal 
positive control target not only provides an indication of general 
amplification performance for a sample (ie., a control for false 
negatives), it also provides a standard for quantitating the initial 
amount of target in the sample. 
EXAMPLE 1 
An IS6110 target sequence of Mycobacterium tuberculosis was amplified by 
tSDA, with inclusion of a signal primer for detection of amplification by 
generation of secondary amplification products. All oligodeoxynucleotides 
were synthesized using standard techniques and purified by gel 
electrophoresis. The 5'-fluorescein labeled signal primer was prepared 
using standard procedures and 6-FAM AMIDITE (Applied Biosystems, Inc.). 
The signal primer hybridized to nucleotide positions 985-1010 of the IS61 
10 element (D. Thierry, et al. 1990. Nucl. Acids Res. 18, 188) and had the 
following sequence: 
5'-ATCCGTATGGTGGATAACGTCTTTCA (SEQ ID NO:1) 
The amplification and bumper primers were as follows, with the BsoBI 
recognition sequence shown in bold italics and the IS6 1 10 target binding 
sequence underlined: 
5'-CGATTCCGCTCCAGACTTCT CTACTGAGATCCCCT (SEQ ID NO:2, S.sub.1) 
5'-ACCGCATCGAATGCATCTCTC AAGGCGTACTCGACC (SEQ ID NO:3, S.sub.2) 
5'-CGCTGAACCGGAT (SEQ ID NO:4, B.sub.1) 
5'-TCCACCCGCCAAC (SEQ ID NO:5, B.sub.2) 
tSDA was performed in 100 .mu.L samples with the final concentrations of 
reagents as follows: 35 mM K.sub.2 HPO.sub.4 (pH 7.5), 3 mM TRIS-HCl (pH 
7.9), 15 mM NaCl, 0.3 mM DTT, 10.5 mM MgCl.sub.2, 1.4 mM each dGTP, dATP, 
TTP and dCTP.alpha.S, 0.1 mg/mL bovine serum albumin, 500 ng human 
placental DNA, 15 nM primer S.sub.1, 6 nM primer S.sub.2, 5 nM each 
primers B.sub.1 and B.sub.2, 320 units BsoBI (New England Biolabs), 8 
units Bca (Panvera), 5 nM 5'-fluorescein labeled signal primer and the 
amounts of M. tuberculosis DNA indicated in Table I. The samples were 
initially prepared in 70 .mu.L of 50 mM K.sub.2 HPO.sub.4 (pH 7.5), 10.7 
mM MgCl.sub.2, 2 mM each dGTP, dATP, TTP and dCTP.alpha.S, 0.14 mg/mL 
bovine serum albumin, 21.4 nM primer S.sub.1, 85.7 nM primer S.sub.2, 7.1 
mM each primers B.sub.1 and B.sub.2, and 7.1 nM 5'-fluorescein labeled 
signal primer. Varying amounts of target were then added to each sample in 
a 10 .mu.L aliquot of 10 mM TRIS-HCL pH 7.9, 10 mM MgCl.sub.2, 50 mM NaCl, 
1 mM DTT with 500 ng of human placental DNA. These 80 .mu.L samples were 
denatured by heating for 2 min. in a boiling water bath and equilibrated 
for 3 min. at 60.degree. C. for primer annealing. BsoBI and exo.sup.- Bca 
polymerase were diluted together to 16 units/1 .mu.L and 0.4 units/.mu.L, 
respectively, in 10 mM TRIS-HCl pH 7.9, 10 mM MgCl.sub.2, 50 mM NaCl, 1 mM 
DTT and added in a 20 .mu.L aliquot to each 80 .mu.L SDA sample 
equilibrated at 60.degree. C. After mixing, SDA was allowed to proceed for 
15 min. at 60.degree. C. and was then terminated by addition of 6 .mu.L of 
0.5M EDTA. The samples were diluted with 0.9 mL of 55 mM NaCl, 111 mM 
TRIS-HCl (pH 7.5), 0.7 mM K.sub.2 HPO.sub.4 (pH 7.4), 1.1 mM EDTA, 0.7 mM 
.beta.-mercaptoethanol, 27 .mu.g/mL bovine serum albumin, 0.02% TRITON 
X-100, 7% (v/v) glycerol. Fluorescence polarization was measured on an 
fluorometer specifically designed for fluorescein (Model FPM-1, Jolley 
Consulting and Research) after equilibration at 37.degree. C. A 
preparation of an exonuclease deficient Kienow fragment of E. coli 
polymerase I (United States Biochemical) was then added (5 .mu.L of a 5 
units/.mu.L stock solution) and fluorescence polarization was recorded a 
second time at 37.degree. C. 
The signal primer exhibited a target amplification-dependent increase in 
fluorescence polarization as shown in Table I (mP): 
TABLE I 
______________________________________ 
Number of M. tuberculosis genomes 
1000 100 10 1 0 
______________________________________ 
114 (154) 108 (136) 79 (95) 62 (68) 57 (60) 
______________________________________ 
*values in parentheses are post polymerase addition 
Samples containing higher input target exhibited higher polarization 
values, while the negative control (0 input target) exhibited a 
polarization value comparable to that of the single-stranded signal 
primer. Amplification of ten M. tuberculosis genomes was clearly 
detectable over the negative control and amplification of one genome was 
slightly increased above background. 
Upon addition of exo.sup.- Klenow polymerase, FP values increased 
considerably for the amplified samples which contained M. tuberculosis 
DNA, resulting in increased assay sensitivity. This is presumably because 
binding of the polymerase to the double-stranded secondary amplification 
product further slows the tumbling time of the fluorescent label on the 
signal primer. There was essentially no increase in FP with added 
polymerase in the sample which did not contain the target, demonstrating 
that there is very little mispriming by the signal primer in tSDA. The 
higher operating temperature of tSDA also reduces non-specific background 
amplification to a level which eliminates the need for 
sequence-specificity in the DNA binding protein. That is, at the 
completion of tSDA the double-stranded DNA present is predominantly 
target-specific, allowing use of a double-stranded DNA binding protein 
which does not specifically bind to the secondary amplification products 
to enhance the change in FP. Any sequence non-specific double-stranded DNA 
binding protein should therefore be effective to enhance the change in FP 
under the conditions of tSDA and other thermophilic amplification 
reactions. 
Evidence of enhancement of the change in FP was evident even before 
addition of the exo.sup.- Klenow polymerase. Similar effects were observed 
in mock SDA reactions where the signal primer was hybridized to a 
complementary oligodeoxynucleotide. In the absence of BsoBI and Bca, FP 
increased from about 55 to 70 mP upon hybridization. Addition of BsoBI and 
Bca resulted in a hybridization-associated increase in FP to about 125 mP. 
These results were unexpected because conventional SDA, with FP similarly 
measured at 37.degree. C., did not show any enhancement in the absence of 
an additional, sequence-specific double-stranded DNA binding protein. The 
results of the mock SDA reactions, and the observation of MP values 
greater than 70 mP prior to addition of polymerase in the high target 
samples in Table I, suggest that double-stranded DNA binding proteins 
present in the amplification reaction also serve to enhance the change in 
FP. Further, fluorescence polarization begins to decrease if tSDA is 
extended beyond the time of maximum target amplification (generally about 
15 min.). This is also the point of the amplification reaction at which 
non-specific background products begin to increase. 
EXAMPLE 2 
Endpoint measurements of fluorescence polarization were used to detect 
amplification of a target sequence in Chlamydia trachomatis elementary 
bodies (EB). EB's of C. trachomatis (serovar E) were heated for 15 min. at 
95.degree. C. to decrease the infectivity of the sample and to lyse the 
EB's. This released the DNA for amplification. Serial dilutions of the 
lysate were made with 10 ng/.mu.L human placental DNA in water. The 
S.sub.1, S.sub.2, B.sub.1 and B.sub.2 primers were synthesized using 
phosphoramidite chemistry on an Applied Biosystems 380B synthesizer as 
recommended by the manufacturer. The oligonoucleotides were ammonium 
hydroxide deprotected at 50.degree. C. for 16 hrs. and purified by gel 
electrophoresis using conventional methods. The signal primer was 
synthesized with a six carbon AMINOLINK (ABI) on the 5' phosphate. This 
oligonucleotide was conjugated to 5-DTAF at its 5' end as follows. The 
amino oligonucleotide (56 .mu.L of a 150 .mu.M solution) was mixed with 60 
.mu.L of 10 NaHCO.sub.3 /Na.sub.2 CO.sub.3 buffer (25 mM, pH 9). To this 
solution was added 10 .mu.L of 40 mM 5-DTAF in DMF. The reaction was 
allowed to incubate at 37.degree. C. for 72 hrs. in the dark. The labeled 
oligonucleotide was first purified from excess unreacted dye by column 
chromatography on a NAP-5 column (Pharmacia) equilibrated with 25 mM 
NaHCO.sub.3 /Na.sub.2 CO.sub.3 buffer. Several 0.5 mL fractions were 
collected and the labeled oligonucleotide was found in Fraction 2. 
Fraction 2 was then further purified to separate labeled from unlabeled 
oligonucleotide using an Oligonucleotide Purification Cartridge (OPN, ABI) 
and conventional protocols. The final fraction was assayed for spectral 
purity on an HP 89532A spectrophotometer, scanning 240-600 nm. Optical 
densities were: A.sub.260 0.11273, A.sub.494 0.0215, A.sub.2601280 1.62, 
A.sub.260/494 5.25. 
For the endpoint measurements, tSDA was performed in a 50 .mu.L volume 
containing 5 mM MgCl.sub.2, 0.2 mM each dGTP, dATP, TTP, 1.4 mM 
dCTP.alpha.S, 20 .mu.g/mL non-acetylated bovine serum albumin, 1 ng/.mu.L 
human placental DNA, 40 mM K.sub.2 HPO.sub.4 pH 7.6, 5% (v/v) glycerol, 3% 
(v/v) DMSO, 750 nM primer S.sub.1, 188 mM primer S.sub.2, 75 mM primers 
B.sub.1 and B.sub.2, 10 nM signal primer, 3.2 units/.mu.L BsoBI, 0.25 
units/.mu.L exonuclease deficient Bst DNA polymerase (Molecular Biology 
Resources) and varying amounts of Chlamydia EB's. To facilitate signal 
primer hybridization to the target prior to hybridization of S.sub.2, in 
this example the S.sub.2 primer was in four-fold lower concentration than 
the S.sub.1 primer and in 18.75-fold higher concentration than the signal 
primer. Prior to addition of BsoBI, Bst polymerase, BSA and MgCl.sub.2, 5 
.mu.L of the target preparation was added and the reactions were heated at 
95.degree. C. for 2 min. to denature the target DNA. This was followed by 
equilibration for 5 min. at 53.5.degree. C. to anneal the primers. 
Following addition of 10 .mu.L of enzyme mix containing 5 .mu.L 50 mM 
MgCl.sub.2, 1 .mu.L BSA (1 mg/mL), 1 .mu.L Bst polymerase (25 
units/.mu.L), 1 .mu.L BsoBI (160 units/.mu.L) and 2 .mu.L 1X NEB2 (10 mM 
TRIS-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl.sub.2, 1 mM dithiothreitol--New 
England BioLabs), the reactions were incubated at 53.5.degree. C. for 30 
min. for amplification. An aliquot of the amplification reaction (45 
.mu.L) was added to 1 mL FP buffer (100 mM TRIS pH7.5, 1 mM EDTA, 44 mM 
NaCl, 0.5 mM .beta.ME, 20 .mu.g/mL BSA, 0.015% TRITON-X 100, 5% glycerol) 
at 37.degree. C. and read in an FPM-1 fluorometer using the following 
settings: read mode--static; single blank, blank delay and sample 
delay--10 sec.; PMT voltage --80; FP Factor--1.0020; lamp feedback--off, 
number read cycles--1. 
The endpoint measurements were used to evaluate the sensitivity of the DTAF 
system, detecting amplification of 500, 50, 10, 5, and 0 EB's. 
Amplification of five Chlamydia EB's could easily be seen above background 
(FIG. 1). Two negative control reactions were run--one without target DNA 
and one in which SDA was inhibited by EDTA. The reaction without target 
showed a slight rise in mP due to contamination with target or amplicons 
from previous reactions at a level less than five EB's. Contamination was 
possible in this system because the endpoint assay was not a closed-tube 
homogeneous assay. As endpoint FP measurements reflect the amount of 
signal primer converted to the double-stranded form, at higher input 
target levels the results are non-quantitative because essentially 100% of 
the signal primer will be converted. While more signal primer could be 
added to the reaction to quantitate higher target concentrations, this 
would compromise sensitivity at lower concentrations of target where more 
signal primer would have to be converted to produce a detectable increase 
in the average correlation time. Again, enhanced FP values (greater than 
about 230 mP using a 5-DTAF end-label) were observed in samples containing 
target, indicating that under thermophilic amplification conditions 
sequence non-specific double-stranded DNA binding proteins present in the 
amplification reaction contribute to enhanced .DELTA.mP by binding to 
secondary amplification products. 
EXAMPLE 3 
tSDA reactions for real-time monitoring were performed as in Example 2 in 1 
mL volumes with 100 .mu.L of the target preparation. There were four 
reactions (containing either 10.sup.6, 10.sup.4, 10.sup.2 or 0 EB's) to 
evaluate the quantitative aspects of the assay. The negative SDA sample 
contained 10 mM EDTA and served as a fluorescence polarization control. 
After denaturing the target at 95.degree. C. for 5 min. the samples (800 
.mu.L) were transferred to a 10 mm.times.45 mm cuvette in an SLM 8100 
spectrofluorometer (Milton-Roy) and equilibrated at 53.5.degree. C. for 10 
min. using water circulating temperature control. Amplification was 
initiated by addition of 200 .mu.L enzyme mix and fluorescence 
polarization was monitored every 2 min. for a total of 45 min. The SLM 
8100 spectrofluorometer has four cuvette chambers which can be read 
sequentially to allow all samples to be analyzed at the same time. 
The polymerase used in the amplification reaction unexpectedly made it 
possible to perform real time detection in a closed-tube format at the 
elevated temperature. End fraying and/or breathing in the double-stranded 
secondary amplification products was apparently reduced or eliminated by 
binding of this double-stranded DNA binding protein, resulting in an 
increase of FP to approximately 220 mP with single- to double-stranded 
conversion of the signal primer. Preliminary experiments had indicated 
that there would be no difference in FP for the single- to double-stranded 
conversion due to inicreased single-strandedness near the label at higher 
temperatures. Although the restriction endonuclease in the amplification 
reaction is also a double-stranded DNA binding protein, Applicants believe 
its contribution to stabilization of the duplex at higher temperatures is 
minimal, as no binding was detected in the absence of a recognition site 
for the restriction endonuclease in the secondary amplification product. 
FIG. 2 shows the results for the first 30 min. of real-time detection of 
target amplification. The reactions containing 10.sup.6 and 10.sup.4 EB's 
plateaued at about 20 min., with mP values of about 200-220. However, a 
quantitative difference could be seen at all target levels during 
real-time detection, as samples containing higher initial levels of target 
showed an increase in FP sooner than those containing lower initial target 
levels. As shown in FIG. 2, 10.sup.6 EB's were detectable by an increase 
in FP in about 8 min., 10.sup.4 EB's were detectable by an increase in FP 
in about 10 min. and 10.sup.2 EB's were detectable by an increase in FP in 
about 16 min. Real-time detection therefore overcomes the limitations of 
endpoint analysis when it is desired to quantitate the initial amount of 
target present. In controlled experiments such as this one, all samples 
are expected to exhibit essentially the same rate of SDA and the assay is 
quantitative. However, clinical samples may contain inhibitors of 
amplification which decrease the rate of amplification relative to 
controls. For real-time quantitation in such samples, an internal control 
sequence at a known initial concentration could be co-amplified in the 
clinical specimen using a different fluorophore, or in a parallel reaction 
using the same fluorophore. The effect of the clinical sample on 
quantitation of the internal control could then be applied to accurately 
quantify the clinical target. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATCCGTATGGTGGATAACGTCTTTCA26 
(2) INFORMATION FOR SEQ ID NO:2: 
(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:2: 
CGATTCCGCTCCAGACTTCTCGGGTCTACTGAGATCCCCT40 
(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: 
ACCGCATCGAATGCATCTCTCGGGTAAGGCGTACTCGACC40 
(2) INFORMATION FOR SEQ ID NO:4: 
(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:4: 
CGCTGAACCGGAT13 
(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: 
TCCACCCGCCAAC13 
__________________________________________________________________________