CA2133643A1 - Method and device for detection of nucleic acid or analyte using total internal reflectance - Google Patents

Abstract

An apparatus and method for detecting amplified target nucleic acid is provided wherein the presence and concentration of amplified target is determined by total internal reflection over the course of the amplification reaction. A method and apparatus for detecting target nucleic acid is also provided wherein the presence and concentration of target is determined by total internal reflection and coupling of the target to the TIR element by scissile linkage. An improved immunoassay using total internal reflection and differential temperature cycling is further provided.

Description

; 2133643 WO 93t20240 ~Cr/US93/03256 METHOD AND DEVICE FOR DETECTION OF NUCLEIC ACID OR
~NALYTE USING TOTAL INTERNAL REFLECTANCE

FIELD OF THE INY~NTION
The present invention rela~es to methods, app~ratus, and kits ~or amplifying and/or detecting target nucleic acid using total internal reflection ("l'rR") techniques. The invention also relates to an improved TIR device and method for specific binding assays, 5 including irnmunoassays.

BACKGROUND DESCRIYrION
The amplification of nucleic acids is useful in a variety of applications. For exarnple, nucleic acid amplification methods have been used in the identification of 10 gene~ic disorders such as sickle-cell anem~a and cysac fibrosis, in detecting the presence of infectious org,anisms, and in typing and quantification of DNA and RNA for cloning and sequencing.
Methods of amplifying nucleic acid sequences are known in the aTt. One method, known a$ the polymerase Ghain reaction ("PCR"), utilizes a pair of oligonucleotide 15 sequences called "p~}mers" and thermal cycling technigues wherein one cycle of denatura-tion, annealing, and prirner extension results in a doubling of ~e target nucleic acid of interest. PCE2. amplification is described further in U.S. Patent Nos. 4,683,195 and 4,6837202, which are incorporated herein by reference.
Another method of amplifying nucleic acid sequences known in the art is the

2 0 ligase chain reac~on ("LCR"). l,ike PCR, LCR utilizes thermal cycling techniques. In LCR, however, two primary probes and two secondary probes are employed instead of `the primer pairs used in PCR. By repeated cycles of hybridization ~nd ligation,a~nplification of the target is achieved. The ligated arnplification products are functionally equivalent to either the target nueleic acid or itS complement. This technique is descTibed 25 ` m~re completely in EP-A-320 308 and EP-A-439 182.
Other methods of amplifying nucleic acids known in `the art involve isotheImal reactions, including the reaction referred to as Q-beta ("Q~") amplification [~, for example, Kramer et al., U.S. Patent No. 4,786,600, WO 91/04340, Cahill et al., Clin.
Chem., 37:1482-1485 (1991); Pritcbard et al., Ann. Biol. Clin., 48:492-497 (1990)~.
30 Ano~er isothermal reaction is descnbed in Walker,et al., "Isothermal in vitroamplifica~ion of DNA ~by a restriction enzyme/DNA polymerase system", Proc.
Natl.Acad. Sci.i. 89:~92-396 (1992). These amplification reactions do not require thermal cycling.

WO 93/20240 ~ 1 ~ 3 6 4 3 PCr/US93/03256 Ampliflcation of nucleic acids using such methods is usually perfoImed in a closed reaction vessel such as a snap-top vial. After the amplification, the reaction vessel is then opened and the amplified product is transferred to a detection apparatus where standard detection methodologies are used.
ln some cases, the a,mplified product is detected by denaturing the double-stranded amplification products, and ~eating those prod~ucts with one or more hybridizing probes having a detectable label. The unhybndizec~robes are typically separated from the hybridized probe, requiring an ex~a separation step. Altematively, the primer or probes may be labeled with a hapten as a reporter group. Following asnplification, the 10 hapten, which has been incorporated into the amplification product, can be used for separation and/or detec~on.
- Ln yet another detection method, the ampli~lcation products may be detected by gels stained with ethidium bromide. ln sum, 32p ~acings, enzyme immunoassay [Keller et al., J. Clin. Microbiolog~, 28:1411-6 (1990)], fluorescence ~Urdea e~ al., Nucleic Acids Research, 16:4937-56 (1988); Smith et al., Nucleic Agds Research, 13:2399-412 (1985)], and che~ilum~nescence assays and the like can be performed to detect nucleic acids in a heterogeneous manner ~Bornstein and ~oyta, (~lin. Chem., 35 :1856-57 (1989); ~3ornstein et al., Anal. Bioch~, 180:95-98 (1989); Tizard et al., Proç. Natl.
A~asL~, 78:4515-18 (1990)] or homogeneo~ls manner [Arnold et al., U.~. Patent No. 4,950,613; Arnold et al., (:~lin. Chem., 35: 1588-1589 (1989); Nelson and Kacian, linica Chimica A~, 194:73-90 (1990)~.
In each case, however, these detection procedures have senous disadvantages.
First7 when the reacuon vessel containing a relatively high concentration of the amplified product is opened, a splash o~r aerosoI is usually foImed. Such a splash or aerosol can be sources of potential contamination, and con~an~ina~on of negative, or not-yet amplified, nucleic acids is a senous probl~m and may lead to erroneous results.
Sirnilar problems concernmg contamination may involve the work areas and equipment used for sample preparation, preparation of the reac~aon reagents, plification, and analysis of the reaction pr~ducts. Such con~nination rnay also occur through cont~t transfer (carryover), or by aerosol generation.
Furthermore, these previously des~ibed detection procedures are time-cons~ming .
and la~or intensive. In the case of both hybridization probes and hapten detection, the arnplification reaction vessel must be opened aIld ~e contents trans~elred to another vessel, medium or ins~ument. Such an "open" detec~ion system is disadvantageous as it

3 5 ~ ~ leads to fu~er contam~na~on problems, both airborne and carryover.
Thus, a need emerges for detec~ing amplified nucleic acids in a closed system in ~ ~ , , ~

WO ~312~)240 ~ 1 3 3 6 4 3 PCI/US~3/03256 order to elimirlate the potential for contamination. Also, a need emerges for a method of amplîfying and detecting the target nucleic acid in an operationally simple, yet highly sensitive manner. The ability to detect the amplification product in a sealed vessel, or in a closed system, offers useful advantages over existing prior art methods, including the ability to mon~tor the ampli~ication of target nucleic acid throughout the course of ~}e reaction.
The use of lotal inte~al reflection fluorescence techniques is known in the ar~
with respect to immunoassays ~Harrick, et al., Anal. Chem., 45:687 (1973)]. Devices and meth~ls that use total internal reflection fluorescence for immunoassays have been described in the ar~ by Hirschfield, U.S. Patent Nos. 4,447,564, 4,577,109, and

4,654,532; Hirschfîeld and Rlock, U.S. Patent Nos. 4,716,121 and 4,582,809, which - are all incorporated herein by Teference. Other descripuons and uses are glven by Glass, U.S. Patent No. 4,844,869; Andarde, U.S. Patent No. 4,368,047; Hirsch~leld, GB
2,190,189A; Laclcie, WO 90/067,229; Block, G~3 2,235,292A, and Carter et al., U.S.
1~ Patent Mo. 4,608,344.
Use of total intenlal reflection elements allows perfon:ning a homogeneous assay(i.e. iree of separation and wash steps) for members of specific binding pairs. SeYeral applications of ~his principle are known in the art [such as Kronick, et al. ~ ~mmur~ol.
Metk~ds, 8:235 ~1975) and U.S. Patent No. 3,604,927] for hapten assays and for immunoassay of macromolecules [Sutherland et al., J. Immunol. Methods, 74:253 (1984)]-In known total internal reflectance methods, however, the slow diffusion of members of specific binding pairs from the bulk of the solutîon to the surface of the TLR
elemcnt ereates a limitation in using TIR fluorescence techniques. Thus, prior art devices have used capillary tu~es or flow cells to enhance diffusion either by limiang the diffusion distances or by con~nuous exposure to fresh reactant s~eam, or both. But thesé systems, too, have drawbacks that make them less than op~mal for clinical biological applications. Capillary tubes are difficult to manipulate and are not easily automated. Flow cells require extensive washing in an effort to reduce caIryov~r3 5 contamina~ion before they can be reused. ~
Thus, in addition ~o a need for contamination-free, closed arnplification systems, ~ere is also a need in ~he art for better TIR assay systems that are more easily automated and even disposable if d-sired.

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' ~ ~

WO 93/~0240 2 1 3 ~ fi 1 3 PC~tUS93/~3256 .

SUMMARY OF THE INVENTION
Several objectives and advantages of the present invention may be stated. First,the invention can monitor the presence and/or conc~-ntration of target molecules in real time. This is particularly of interest in nucleic acid amplification reactions. In addition, it

5 is an object of the present invention to reduce contamination~o~ other samples and other unused vessels and reagents wi~h the amplifled target nucl~e~c acid through the use of a sealed vessel in which both amplification and detection occur. A still further objec~ of the present invention is to provide relatively simple and sensitive methods and apparatus for detecting target nucleic acid or other molecules of interest in a reaction sample.
Accordingly, in a first aspect, the inventon is a method of detecting amplified target nucleic acid using total internal reflection, comprising the steps of:
- providing a reaction vessel having disposed therein (a) a reaction sample, (b~ a total internal reflection (TIR) element, (c) a pl~ality of members of initiator sequence sets and reagents for producing amplification of target nucleic acid present in the reaction 15 sample, (d) label means which is coupled to a fluorophore, and (e) capture means for brhlging said fluorophore within the penetra~ion depth of said element, wherein at least.
one of said label means and said c~Lpture means is specific for said target nucleic acid;
producing an evanescent ele~tromagnetic wave in the ~IR element which penetrates into the reaction sample adjacent the~element and has an associated penetration 20 depth`;
reac~ing the reaction sample, the initiator sequences and amplification reagentsunder conditions sufficient to amplify target nucleic acid present in ~he reaction sample tO
produce amplification products, c aptunng said label means; within the pene~ation depth as a fimc~ion of the 2~ presence or amount of target nucleic acid; and~
detecting~within the~T~ element a change in fluor~scence.
The invention contemplates both covalent attachment and specific binding member at~hm! ent of initiators ~o tbe element to b:ring~the fluorophore within the pene~a~ionl depth. Both immunoreactive and polynucleo~ide specific binding pairs are contemplated.
30 lt Is p~ferred that the ampllfication mioators~double as either capture means or label means or both. ~ ~
The inventlon~also provides arl apparatus ~or amplification and detection nucleic; acid ta~gets compnsing~
a sealed9 static-volumetlic reaction~vessel adapted to contain a reaction sample and 35; reagents for amplification ~ ~
a total inte~nal reflection (TIR) element disposed in said reaction vessel such that ~: :, , ; : : :

~:;
WO 93~0240 ~ 2 1 3 3 ~ ~ 3 P~/US93/032~6 -substantial surface area of the element is in contact with s~ud reaction sample and such ~hat one end of the e}ement protrudes from the vessel;
means for producing an evanescent electromagnetic wave in the TIR element which penetrates into the reaction sample adjacent the element and has an associated penetration depth;
temperature controi means for reacting the reaction sample and amplification reagents under cyclic tempera~e conditions sufficient tO amplify target nucleic acid present in the reaction sample and to capture a fluorophorc capable of emitting fluorescence within the penet~dtion depth of the element as a function of the presence or amount of target in the sample; and means for detecting in the TIR element a change in fluorescence.
Preferably the reaction vessel and the TIR element are separated from one another by a distance that precludes capillary migration. For wettable vessels and aqueous solutions a distance of 1.7 mm or more is sufficient. The reac~on vessel may b~ sealed by a sealing member having a throughbore for ~e T~ element, or by an integral cap/~IlR
element~
In another aspect, the ~nvention r~lates to a rnethod and apparanls for detecting nucleic acid in a sample by means of signal amplification achieved by destroying, as a func~on of the amount of target present, a scissile link that holds fluorophore near the TIR element. Thus, a decrease in the totally internally reflected fluorescence will occur in the presence of target.
In a final aspect, the invention relates to an impro~ed method and apparatus forconducting s~:cific bindil-g assays with fluorophore labels that a~e detected or monitored by total inte~nal refleG~ance means. This embodiment of the inven~on includes:
a sealed, static-volumetric reaction vessel adapted to contain a reac~on sample and reagents for a specific binding assay; and a total int~nal reflection CIIR) element disposed in said rçaction vessel such that substan~ial surface area of the element is in contact with said reaction sample and such that vne end of the element prvtrudes from the vessel;
wherein said static-volumetric reaction vessel and TIR element are dimensioned such tha~ the space ~etween the element surface an~ the interior wall of ~he reac~on vessel is too great to sup~rt capillary migraqon of an aqueous fluid.
The invenaon also provides kits for detec~ing amplified nucleic acids, comprising PCR or LCR amplification reagents and a TIR element having a plurality of coupling sites that allow attachment of amplified target nucleic acid.

~2 ~ 33~
WO 93/20~40 ~ PCr/U~93/~32~6 BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic illustration of a detection system in accordance with one embodiment of the present inven~on.
Figure 2 illustrates the reac~on vessel and associated excitation and detection 5 opncs for a detection system as shown in Figure 1.
Figure 3 illustrates a unitary fused embodiment cornprising a cylindrical total internal reflection element, a lens therefor,and a reactlon chamber seal Figure 4 illustrates a unitary fused embodiment comprising a planar or flat total internal reflection element, a beveled pnsmatic lens therefor,and a reaction charnber seal Figure SA illustrates a preferred configura~ion for PCR amplification, using a capture initiator and a label initiator.
~ igure SE~ illustrates a preferred configuration for LCR amplification, using capture initiator and label initiator probe pairs.
DETAILED DESCP~IPTIC)N
15 A . TIP~ Principles:
Total internal reflection ("TIR") is known in the art and operates upon the principle that light striking ~he interface between two media having different refractive indices (Nl>N2) from the denser medium (i.e. having the higher refracave index; here Nl) can be made to totally internally reflect witl~in the medium if it strikes the interface at an angle, ~R, greater than the critical angle, ~ C, where the critical angle is defimed by the equation:

~ C = arcsin (N2/Nl) Under these conditions, an electromagnetic waveform known as an evanescerlt wave is generated i1 ~he less dense medium, and the electriG field associated with the excitation llght ~orms a standing sinusoidal wave, normal to the interface, is established in the denser medium. The evanescent wave penetrates into the less dense medium, but its jen~rgy dissipates exponenti:ally as a function of distance from the interface. A
parameter known as S'penetration depth" (dp) is defined as the distance from the interface at which the evanescent wave energy has fallen to 0.368 times the energy value at the interface. [~, Sutherland et al., J. Immunol. Meth., 74:253-265 (1984)J. Penetration dep~ is calcul~ted as ~lIows:

~/N1 d , 1. ~ 1 3 3 6 4 3 WO g3/20~40 Pcrlus93tO32~6 Factors that tend to increase the penetration depth are: increasing angle of incidenee, ~R; closely matching Indices of refraction of the two media (i.e.
N2/NI --> l); and increasing wavelength, ~. An example will illustrate. If a quartz TIR
element (N1 ~ 1.46) is placed in a aqueous medium (N2 = 1.34), the critical angle, a C, is 66. If SOO nm light impacts the interface at ~R ~ 70 (i.e. greater than the critical angle) the dp is approximately 150 nm.
For cylindr:.cal and fiber optic TIR elements, the maxirnum acceptance angle with regard to the TIR element axis, B, for the radianon entering the TIR element and so propagated within it, is established by the refractive indices of the TIE~ element and the surrounding medium. For radiation initially propagating through a medium of refractive index No, such as a~r, incident ~lpon a TIR element of refractive index N1, otheIwise - surrounded by a medium of rcfrac~ve index N~, the maximum acceptance angle, B, may be ~ound from the equation:
N.A. = No sin B = (N12 - N22)1/2, where N.A. is the so-called numerical aperture of ~e TIR element.
Within the penetration depth, the evanescent wave in the less dense medium (typically a reaction solution) can excite fluorescence in the sample. The fluorescence Nnnels back into the TIR element propagates within the TIR element ~long the same path as the standing sinusoidal wave (but at a different wavelength) and is detected. All of the radia~aon that nmnels back into ~e TI~ element is within the total intemal reflection angle and is thus ~apped within the TIR element. Accordingly, llR allows detection of a fluorophore-labeled target of interest as a function of ~e amount of the ~arget in the reaction sample that is within the pene~ation depth of the TIE~ element.

B. ~ A ~irst Ernbodiment:
In accordance with a first embodiment of the present invention, total internal reflec~on is used to detect amplifled target nucleic acid in a reaction vessel. The reacaon vessel preferably is sea}ed although a flow cell or a capillary tube may be used. Both amplihcation ~nd detection can ~take place within the sarne closed reaction vessel, thus minimizing contarr~ination risks.
Figure l illustrates a an amplification and detection system 10 in accordance with one embodiment of the ~present invention. The system includes a thennal cycling device generally represented as 12~ a reaction unit generally represented as 14, fluorescence excita~on source and optics 16 and fluorescence detection op~cs 18. The unit 14 includes a reaction vessel 20,~ a sealing mem~er 22 and a total internal reflection (TIR) ~: ' WO 93t2û240 - P~r/US93/0~56 element 24. The reaction vessel 20 is placed in a thennal cycling device 12 and is suppolted by tab members 26.
Amplification reactions using thermal cycling are presently prefe~ed over isothennal mechanisms. It is believed that convection currents resulting from the suecessive heating and cooling cycles during`th`ermal cycling enhances diffusion of molecules in the reaction sample, although (in contrast to an embodiinent described later) tnis feature is not deerned essential to this embodiment. Accordingly, a thermocycler device 12 is shown. However, the details of t'ne method of thermocycling are not critical to tne inven~on. For example, the temperature of tne amplification reaetion may be 1 0 controlled manually, such as by air or water baths, or regulated automatically by a thermocycler device specifically designed for nucleic acid amplifica~ion. Thennocycler - devices are commercially available from Perkin-Elmer Corpora~ion, (NorwaLtc, CI`) and Coy Laboratories, (Ann Arbort MI).
The reaction vessel 20 is made of glass or polymenc materials such as polystyrene, polyacrylate and the like, and is preferably made of a the~nostable material.
Preferably, the siæ of t'ne reaction vessel 20 is selected so as to contain relatively S7L~Ia11 quantities of reaction sample. More preferably, the reaction vessel 20 is selected to so as to contain from about 50 ul to about 200 ul reaction sarnple. In a typical embodiment, the reaction vessel 20 is a microcentrifuge tube, although other configura2ions are possible and within the inven~on. As will be described (and defined) in more detail below in connection with another embodiment7 it is preferred that the reaction vessel be a "static-volumetric" vessel, having a composition (with regard to wettability) and a distance between the elernent surface 38 Lmd the walls of the reaction vessel 20 that is suf~lciently great to prohibit capillary ac~ion of an aqueous sample therebetween.
The TIR element 24 may be preferably any vf a number of optically transpairent materials, including but not limited to, glass, quartz, and transparent polymers such as polystyrene or polystyrene copolymers and polyacrylic acids or the like, chosen to have an index of refraction greater than thiat of the medium in which it is placed. Preferably the mediurn is an aqueous reacdon sample comprising ampli~lcation reaction reagents and 3 0 target nucleic acids. Such a reac~ion medium typically will have a refractive index ranging from about 1.30 ~o about 1.38, more typically, about 1.34. Thus, for a visible light beam having a wavelength ranging from about 480 to 540 nm, the preferred TIR
elements according to the invention have re~ractive indices ranging f~m about 1.4 1o 1.6.
Exemplary materials and their approximate refractive indices are given in Table 1 below:

:::

WO 93/20240 PCT/US93~03~5S

Table 1 Element Material Refrac~ve Index Quartz 1.6 Polystyrene 1.59 Glass 1.52 Polymethylmethacrylate1.49 ~ ~-- .
The T~R: element 24 is further chosen to be insoluble and non-reactive with the reaction sample. An exemplary TIR element 24 is a glass rod with a wide surface area and a diameter of apploximately 1 rnillimeter. It will be understood that the dimensions 5 of the TIR element 24 accommodate the reac~on being undsrtal~en and the size of the ~eaction vessel 20. Those skilled in the aIt will appreciate tha~ the surface area of the TIR
element 24 should be considered and it is believed ~hat to obtain maximum surface area binding, the reaction vessel 20 and the IIR elemant 24 are preferably long and cylindrical.
10 ~ As shown in figure 1, a sealing member 22 is configured and dimensioned to fit on the open end of the reaction vessel 20. A cent~ally disposed bore 3û in the sealing member 22 is adapted to sup~rt an upper end por~on of the TIR element 24 substantially coaxially within the vessel 20. Additionally, the sealing member ~2 preferably provides a st~rdy l~ating surface ~e.g. tab rnembers 26) for posiaoning the unit 14 with respect to 15 ~the excitation source and optics 16 and detection optics 18, which will be described in more detail in coMec~ion with figure 2. llle sealing member 22 is preferably a rubber sçptum:or a polymer or polymer laminate. : ~ ~
The TI~ element 24 passes through and is supported by the sealing member 22 so as to expose as~much as~possible of the TIR element 24~ to the interior of the reac~on 20 ~ vessèl 2Q, leaving only an end face 32 unobscur~d and approximately coterm~nous with the extremi~y of the bore 30 external to the vessel 20. The end face 32 of the TIR element 24, however, does not hàve to be coteIminous with the extremity of the bore 30 as can be seen from alternative IIR elements shown in filgures 3 and 4. It is imp~rtan~, however, t hat a~minimum amount of ~the TIR element is exposed above the sealing member 22 to 25 ~ reduce the dissipa~on o~ signals:received and transmitted by the TfR element 24.
Preferably the~end ~ace 32 is h~ghly transparent and free of blemishes which would tend to sca~ light incident upon its face.~ The end face 32 may be opncally polished, or :: alternadvely, a~filsed quartz:llR element 24 may be cleaved to provide an adequate op~cal su~face. Other TIR element configuranons will be des~ribed with reference to 2~33~3 ,:;;
~VO 93/2024~) - PC~ 93/03256 Figures 3 and 4.
Alternatively, the TIR element may be fabricated by injection molding of chemically activated transparent polymers into ~ àppropriate shape and finish.
Chemically-activated transparent polymers inclù`~e surface treated homopolymers (e.g.
5 polystyrene), and copolymers of styrene such as styrene maleic anhydride (commercially available from ARCO Chemical Company). It is very likely that other polymers andcopolymers are suitable provided they are transparent and chemically activatable.
In the embodiment of Figure 1, opposite end face 34 of the TIR element 24 is also polished Mat or cleaved and, preferably, is further provided with a black coating, a mirror 10 coat~ng or a separate m~rror disposed substantially normal to the TIR element 24 axis. It is impo~t in the operation of the invention to avoid fluorescent excitation of the buLk reaction solution by light exiting the TIR element 24 through the end face 34. Thus, a black coating (to absorb) or a rnirror coating (to re~lect) are preferred. The rnirror coa~ang or separate mirror has the added advantage of causing radiation trapped in the TIR
element 24 to double pass the TIR element 24. The end face 34 need not be flat or no~nal to the axis of the element, however, as shown from figure 3.
In the interior of the re~ction vessel 20, the TIR elemen~ 24 is exposed to the reaction sample 36. The reac~on sample 36 typically mcludes a buffered solution of sample components, label means and reagents for amplification (descnbed further 20 below). Examples of typical reaction sarnples for particular ampli~lca~on reac~ons are provided in Examples 4-11 below.
The outer surface 38 of the TIR element 24 is modified, as described further below, having a plurality of coupling sites that allow attachment of the amplification reac~ion products or other members of specific binding pairs tha~ can capture 25 amplificaeion reaction products. The ampli~led product, ~ypically a double-stranded nucleic acid, comprises a pendent fluorophore, as descri'oed more ~ully below. During ~-the course of or after ~he amplifica~ion reaction, the amplified product and associated fluorophore is brought within the penetration depth of the TIR element 24 so that a fluorescent signal may be detected~

30 C. Reagents arld Protocols:
The target nucleic acid of the present inven~on is that nucleic acid sequence sought to be detected. It ~may comprise deoxyribonucleic acid (DNA) or ribs~nucleic acid (RNA), or may be natural or synthetic analogs, ~ragments, and/or deriva~ves thereof The ~get is preferably a na~urally-occurring nucleic acid of prokaryo~ic or eukaryo~c 35 ongin, including but not limited to, human, hurnan immunodeficiency virus (HIV), human papilloma virus (HPV), herpes simplex virus (HSV), Chlamydia, . ~;,. .
, ....

WO 93~202d,û PCr/US93t03256 Mycobacte~ium, Streptococcus, and Neissena. One of skill in the art will recognize that thousands of other target nucleic acid sources are possible. When possible, DNA is often preferred due to its better stability.
As mentioned above, the outer sur~ace 38 of the TIR element 24 is modified to 5 include a plurality of coupling sites for attachment of "capture means" for bringing fluorophore within the penetration depth. Various capture means are described below, and include covalent bonding and specific binding pair attachment. In addition to the "capture means", it is necessary to have a "label means" for absorbing and re-emitting the fluorescent energy. The label means comprises a fluorophore, which is capable of0 absorbing fluorescent energy at one wavelength and re-e~utting energy at a different wavelength, as is known in the art. Either the capture means or the label means, or both, must be specifically associated with the presence or amount of target. The present TIR
invention depends on the ability to bnng the label means within the penetration depth in amounts that correspond to the presence or amount of Ihe target.
A third reagent system is necessary for amplification of the ta~get. Amplification reactions contemplated by the present invention include, but ~re not lin~ited to, thermal cycling reactions such as PCR and LCR~ and isothermal reactions such as Q-beta and restriction/polymerase ~mpliffcation. Other amplification systems yet to be developed may also be useful. Target amplification typically requires a polynucleotide 20 complementary to a region of the target molecule. The tenn "initiator", as used in the present invention, is intended to refer generally to such a polynucleotide which is c~pable of sufficiently hybridizing with the target nucleic acid to commence ~e amplification process. Initiators are selected to be complementary to various por~ons of ~e target nucleic acid. For puIposes of this invention, no distinc~ion is drawn between ~5 "pol~ucleotide" and "oligonucleotide".
The initiator serves different func~ons depending upon the ~pe of amplifica~on reaction employed. In the PCR amplification reaction, the ini~ator (typically re~erred to in~ the art as a primer) acts as a point of hy}7ridization and initia~ion of the enzyrnatic poly~erization step that results in extension. Each initiator is then extended by a i 30 polymerase using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the ori~inal ~arget strand. For LCR amplification~ the~in}tiatMs, (typically referred to in the art as probes) comprise four polynu~leotides, two of which (primary) hybridize to the target strand such that they become ligated togethe~, and two~of which (seconda~y) hybridi~e to the target 35 ~ complement or the ligated pnmary product and are similarly ligated. Both PCR and LCR
are amply describ~d in the aIt and need not be de~ailed here.

- i:
WO 93/~0240 PCr/U~93/032~6 It will be realized that by modifying the initiators, it is possible for the functions of capture and/or label to be accomplished by the initiators themselves, alone or in combination with accessory reagen~s. For example, a P;C~ primer labeled with a hapten at its 5' end might fulfill the function of initiator while thè hapten serves as a capture 5 means. In such a case, the primer is referred to as à "capture initiator". Similarly, an ~CR probe labeled with a fluorophore might fulfill the function of initiator and label means, and is thereby called a "label initiator". Table 2, below, provides several possible configurations and reaction protocols, all of which are within ~he present invention.
In a particularly preferred embodiment, the initiators serve all three functions10 (initiation of amp~ification, capture means and label means). Capture and label initiators ~r PCR are illustTated generally in figure ~A. At the end of several cycles in the presence of target (to serve as template for initial extension), the predominant product is a bihaptenated duplex. Other exarnples of capnlre and label initiators that may be utilized for PCR amplification reactions in the present invention are provided in Table 3 of 15 Exarnple 1 below. Analogously, capture and label initiator pairs for LCR are illustrated generally in Figure SB, although they need not be blunt ended as shown. Arbitrarily, initiator (1) and initiator (3) (referred to in Table 2 as "left probes") are designated capture initiator sequences and initiators (2) and (4) (refelTed to in Table 2 as ~'right probes") are designated label initiator sequences. Once the initiators (l) and (2) are ligated, (an event 20 that is essentially dependent on the presence of earget) the fused product is bihaptenated, bearing a first hapten (Hl) on one end and a second hapten (H2) on the other. One hapten is used for capture and the other is used for labeling. Initiators (2) and (4) rnight also have been labelled directly with a fluorophore~ Other examples of capn~re and label initiators that may be utilized for LC~ in ~he present invention are prov~ded in Table 3 of 2~ Example 1 below.
Preferably, the initiator is synthesized using nucleotide phosphoram~di~e or phosphonate chemistry techniques known in ~he art and/or instrumen s commercially availa jble from Applied Blosystems, Inc. (Foster City, CA), DuPont (Wilmington, DE) or ' ~Iilligen (Bedford, ~A). lnitiator synthesis using such techniques is descri~ed further in 30 Example 1 below. Altematively, ~e initiator may be obtained by digeshng naturally-occu~ing nucleic acids and isolating fragments of interest.

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WO 93/20~4û PCr/U~93/03256 Many different haptens are known, and virtually any hapten may be used with the present invention. Many methods of adding haptens to probes are known in the literature. Enzo Biochemical (New York) and Clontech (Palo Alto) both have described and comrne~cialized probe labelling techniques. Fvr example, a primary amine can be ateached to a 3' oligo end USillg 3'-Amine-ON ÇPGrM (Clontech, Palo Alto, CA).
Similarly, a primary amine can be a~tached to a 5' oligo end using Arninomodifier II(~) (Clontech). The arI~ines can be reacted to various haptens using conventional activation and linking chemistries. Alternatively, certain haptens and labels are commercially available as phosphorarI~idite reagents and can be incorporated directly into initiators dur~ng synthesis.
ln addition, copending applications U.S. Serial Nos. 625,566, filed December - 11, 1990 a~d 630,908, filed December 20, 1990 teach methods for labelling probes at their 5' and 3' ends respectively. Both the aforemen~oned copending applications are incorporated by reference. Some illustrative hap~ens include many drugs (eg. digoxin, theophylline, ~hencyclidine (PCP), salicylate, etc.), T3, biotin, fluorescein (F~C), dansyl, 2,4-dinitrophenol (DNP~; and modified nucleotides such as bromouracil and bases modified by incorporanon of a N-acetyl-7-iodo-2-fluorenylamino ~AIF) group; as ~well as many others. Celtain haptens described herein are disclosed in co-pending, co-owned patent applications U.S. 07/808,508 (adamantaneacetic acid), U.S. 0~/808,839 (carbazole and diben~ofuran), both filed December 17, 1991, U.S. 07/858,929 (acridines~ and U.S. 07/ 858,820 (quinolines), both filed March 27, 1992 (collectively refe~Ted to herein as the "hapten applicaoons"). The en~re disclosure of each of the above hapten applications is Incorporated herein by reference.
It will be apparent ~o those persons skilled in the art that, when initiators are labeled on the exterior ends, the length of the amplification products, including any spacers or specific binding partners, should not exceed the penetration depth. Otherwise, the fluorophore label will not become excited. Relatively short amplified targets are produ~ed in LCR so that, even when end-labeled, exceeding the pene~ation depth is usually not a factor. For example, two 25-mer initiators ligated together and ~orming ani 3û alpha helix duplex will have a length of about 17 nm. Even with allowances for spacers ~; and conJugation partners, this is well within the typical 150 nm penetration depth (see above). In contrast, PCR typically amplifies longer target nucleic acid sequences to generate amplification products having from about 100 to several thousand or more nucleotides, including sequences not complementary to the initiator. I hus, ~or PCR it is preferable to select pnrneTs that are relatively close together, or to label the amplification pIOdUGt at intPrnal positions, away ~om the ex~eme ends.

~:
~, C~ 133 6 ~3 ~ PCI /US93~03256 WO g3/20240 The length of the Initiator sequence will also depend on various factors, including but not limited to, arnplification reaction temperature, source of the initiator sequence, complexity of the target sequence, and the method of amplification. Preferably, the initiator sequence is sufficiently long to provide desired specificity in order to avo1d 5 hybndization with random sequences that may be present in the reaction sample.However, particularly with PCR the specificity can be improved using a specific capture o r label probe internal to the pnmers. Preferably, the initiator sequence compnses from about 15 to about 100 bases, and more preferably, from about 15 to about 40 bases.
The amount of in~tiator added to the reaction sample or, in the case of bound 10 capture initiators, coupled to the TIR element, may~be determined empincally by those ~persons skilled in the art. ~Generally, the~amount of initiator added will be similar to that - typically used m nucleic acid ampllficanon reac~ons, i.e. a molar~ excess of about 108 to ; 1012 over the anticipated amount of target nucleic acid in the reaction sample ~which inevitably is unknown in the first place). When covalently attaching bound capture 15~ initiuors or bound components of capture means ~e.g. an~-hapten or complementa~y polynucleodde) it will generally be des~re~to saturate the elernent as completdy as nte ~ element 24 may~be modified by vaTious means so as to allow attachment of ~he arnplification prodùc~s~or other ~embers :~f specific~ binding pairs that can capture 20 ~ the àmpliflcation products. ~lt wal be apparent w those persons s~lled in the~art that means~f~r;a~aching the~àmplifica~on reaction products to the TIR element should be selected~in view of the~amplificat~ion~reachon conditions. For instance, for an ; arnplific;ldonreaction~thatu~lizestherrnalcycling,the}mostablecoupl~g:mechanisms sùch as~cov~ent linkages or p~lynucleo~de linkages~should be selected, while 25;~;: thernolabileli~àges~sùchas~ànti~-haptenshould~av~ Suchc n~ io s ;~
are less cndcal~f~r isothe~nal processes.~ ~The TI~ element~24 modificaaons descnbed bt re;pr~ided`byw~ofex m~e~da e~n~tint~ded~to~ e~l it~g. ~
At least part bf~the~capture~means is generally coupled to the IIR element 24 bycovalent bonding, al~ugh antib~dies may be adsorbed onto:the el~men~ su~fac!e.
n~ =~We~c~ll U.s.Pa~3,65Z.761. Iilethodsof= g~;
n~ybm g~ n~t s~c e ~Iy-~

Sj~arly,~methods~of covalently bon~ng polynucl~des to the element ~e~so~
35~ known~inthe~t.~ Morép~cul~ly, acapt~epolynucleotide (whetherinitiatoror ` spe~fic ~inding pa ~ ~ay~ co~ to~qu~ ~ glass ~R elements u g, WO ~3/20240 2133fi ~3 PCI/US93/03~56 example, methods described in WO 89/10977 and/or WO 90l03382. Chemical binding of nucleotide base pairs to a glass surface typically involves reacting the hydroxyl moieties of the quartz or ~lass surface with trimethyl siloxane, substituted with a chain of methylene groups and terminating with a reactive orga~nic functional group. Prior art silation reactions ~or derivatizing a glass surface are:further described in GB 2,190,189A. Chemical reagents may also be rèacted with a diisothiocyanate to produce amino, benzyl chloridem or isothiocyanate terminal groups on the derivatized glass surface 38 of TIR element 24. These reactive groups may then aetach the capture polynucleotide (initiator or specific binding member) to the TIR element 24. Reagents for 10 chemical binding of nucleotides to the TIR surface are co~nercially aYailable from companies such as Huls AmeTica, Inc. (Piscataway, NJ~, PCR Inc., (Gainesville, FL) or - Petrarch Chemica} Co., arnong others.
Polynucleotides may also be coupled to chemically-activated polymeric TIR
elements. For instance, styrene maleic anhydride (available from ARCO Chemical 15 Company) comp~ises functional groups that allow coupling of the capture means to the TIR element. Attachment of iniaators tO such chemically-activated TIR elements is further described in Example 2 below. Other rnethods of attaching polynucleotides to polystyrene are described in Rasmussen, et al., Anal. Biochem ~198:138-142 (1991).
Alternatively, capture means (whether initiator or specific b~nd~ng member) may 20 be coupled to tne TIR element 24 via spacer arrn linkers. As used in the present invention, the te~Tn "spacer" or "spacer arrn linker" refers to a molecule that extends the capture means, and thus the captured, amplified target away from the sufface of the TIR
~lement, and that ~oes not absorb fluorescence. One form of a "spacer" is specific binding member, such as an antibody or polynucleotide, used to capture the amplifica~on 25 product. Examples of speclfic binding member pairs include, but are not limited to, an~biotin antibodies, avidin, carbohydrates and lectin, polynucleotides. Another form of "spacer" is a chemical linker such as a heterobifunc~onal linker, or poly(same nucleotide) ;' ! i taiL In general, polynucleotides are preferred spacers smce they are thermostable and encounter less steric constraints and competition for the binding sites. For instance~, poly 30 T spacer arms may be used to attach initiator sequences to TIR elements, as described further in Exa~nples 5, 7, 9, and 11 below. Speci~lc binding pairs, including antibodies, may also be coupled to the TIR elernent using prior art spacer arm chemis~y.
Label means preferably compnse a detectable fluorescent label a~tached to at least one nucleotide or a specific binding partner. Label means are typically added to the 35 reaction solution. It will be recalled that in some cases, it is desired tO have an ini~ator that serves part of the label means function. To make a "dir~ct" label initiator, a : ' ~1 33643 WO ~3f20240 PCr/US93/û32~6 fluorophore is covalently coupled tO the label initiator sequence using standard chemistry techniques known in the art ~See. e.,~., Goodchild, Biocon~gate Chemist~ 165-186(1990); or Urdea, et al., Nucl. Acids Res., 16: 4937-4956 (1988)]. Alternatively, an "indirect" label initiator" can be prepared by haptenating one or more initia~ors with a 5 hapten that is differentiable from any capture hapten that might be used.
In yet ano~her alternative, fluorophore-labeled nucleoside tliphosphates, dATP, dCTP, dl~P, dGTP ~commercially available from e.g. PhaI~nacia-LKB Nuclear, Inc, Gaithersburg, MD) rnay be incorporated into the label initiator during synthesis of the sec~uence. This method is particularly useful for PCR and gap filling l.CR.
10 Fluorophores contemplated by the present invention include, but are not limited to, fluorescein, rhodamine, acndine orange, and Texas red. Such fluorophores are commercially available from Sigma Chemical Company (St. Louis, MO), Aldrich Cher~ical Company (Milwaukee, WI), and Molecular Probes (Junction City, OR).
Intercalating fluoroI)hores may~also be useful in the present invention.
The present inventlon contemplates tha~ single or multiple fluorophores may be coupled to a label means (whether initiator or label conjugate). It is believed that it may be advantageous to couple multiple fluorophores to the label initia~or in order to enhance ., ;~ the~fluo~escent~signal, paracu}arly when the reaction sample is turbid. If multiple fluorophores are coupl to the label initiator, the fluorophores should not interfere with 20 ~ hybridizationi polymerization or ligation. To detect multiple target nucleic acid sequences~ or to detect a single ~ et along with a control nucleic acid sequence, ~wo ~ different fluorophores specific~ to each target may be coupled to respective label initiator ; ~ ~ sequences.
Using standard ch~en~istry techniques k~iown in the art, it is possible to couple a 25 ~ fluorophore (or other label~means)~to the 3' or the 5' te~ninus of a label ini~iator. In PCR
ampli;fication, it is preferab!e to label a label initiator at the 5' hydroxyl group, since the 3' terminus is neéded for extension during amplification. ~ Figure ~A). In LCR, it is pre,ferable to couple the label means-to the distal ~from the element~ 5' and 3' termini of the label initiators. ~ (~ Figure SB). The label means may also be eoupled to the label 30~ ~ ~ initiator~internally, as~long as the intemal coupling does not interfere ~ith hybridization or The fluorophore ~ay be coupled to the label ini~at3r directly through sites present in~the sequencé, such as ~amino groups on the bases,~ hydroxyl groups, and phosphate ~.-roups.~AIternahvely, the~fluorophore may be coupled to the label initiator through some :35 ~ o~er~reactive~ linker group~introduced for that purpose. Common reactive linker groups i nclude~primary amines,~thiols~ M aldehydes. Reactive linker groups may also be Wo93/2o24~ 336~.3 PCI/US93/032~6 attached to the label initiator by a spacer arm either to facilitate coupling or to distance the label ~neans from the initiator. For instance9 a hapten may be attached to the label initiator and the fluorophore may then be coupled to the initiator via anti-hapten-fluoro~hore ' conjugate binding [~, ~, EP-A 357 Oll,and EP-A-439 182}.
It will be readily apparent to those persons skilled in the art that, like capture coupling, fluorophore coupling techniques should be chosen in view of the amplification reaction conditions, some methods being more preferable. For exarnple, if PCR or LCR
amplification is employed, the coupling of t'ne fluorophore or fluorophores should involve t'nermostable bonds since both PCR and LCR requi;e thermal cycling.

10 D. Systern:
Figure 2 illustrates the reaction vessel 20 and, from figure 1, the associated fluorescence excitation source and optics 16 and detection optics 18. The fluorescence excitation source and optics and the detection optics are conventional and well known and, in this regard, are not part of the present invention. For completeness, however, a 15 par~icular configuration of the opncs will be described hereinafter for exemplary purposes only. Many other corlfigurations are possible as is well known to those slcilled in the ar~.
The excitation source and op~cs 16 includes a light source 40 and appropriate beam shaping optics 42, as will be well understood by those sldlled in the art, to pern~it the source 40 to be imaged on the end ~ace 32 of the TIR element 24. The angle of incidence 20 of the ray on the end ~ace 32 of the TIR 32 is within the numencal aperture of the TIR
element 24 and ~eater than the cri~cal angle described above. The appropriate beam shaping optics 42 may include a coll~mating lens 44, an excitation wavelength selection means 46 and a focusing lens 48 as is well known to those skille~ in the art. The light source 40 may be a direct current-driven tungsten-halogen lamp, a phosphor coated 25 mercu~ lamp, a pulsed Xenon flash lamp or a laser. The excitation wavelength selechon means 46 can be a prior art nar~ow band mul~cavity interference filter having a maximum wavelength transmission chosen to allow optimum excita~on of the fluorophore or fluQrophores selec;ted.
The light source 40 and wavelength selection means 46 provide optical radiaaon 30 of the appropriate frequency, chosen on the basis of the fluorophore or flu~rophores employed, to excite fluorescence in the la~el means associated with ampli~led target nucIelc acid. The light source 40 preferably provides this radiation only over a narrow waveleng~h band chosen to maximize fluorescence. Of course, multiple fluorophores may be usedO Alternatively the light source 40 may provide optical radiation of multiple 35 frequencies ~o excite multiple fluorophores. If multiple fluorophores are used, each of the fluorophores is selected so that the absorp~ion maximum of one fluorophore is not ~ 2 1 3 :~ 6 4 3 PCr~US93/03256 WO 93/20~40 near the emission rnaximurn of another fluorophore, and so that the emission wavelengths are distinguishable The fluorescence detection optics 18 includes a detector 50, field optics 52 anddetector electronics 54. The detector 50 is chosen to have maximum sensitivity in the 5 region of peak fluorescence emission of the fluorophore; the field optics 52 restrict the detector's 50 ~leld of view to the end face 32 of the TIR element 24, as is well known by those slcilled in the art. The field optics 52 include a collimating lens 56, a fluorescence wavelength selection means 58 and a focusing lens 60. llle detector 50 can be a photodiode, an avalanche photodiode, or a photomultiplier tube. When using a pulsed 10 light source, time-ga~ed detection can be used to improve the signal to noisecharactenstics of the system. The fluorescence wavelength selection filter 58 is chosen to - maximize transmission of the emission fluorescence bearn(s) and to haYe maximum blocking at other wavelongths, especially the excitation wavelength.
If multiple fluorophores are used, they may be excited at different wavelengths or 15 each of the fluorophores may be excited at the same wavelength, but emit at different wavelengths, provided the absorption maximum of one fluorophore is not near the emission maximum of another fluorophore. If multiple fluorophores are used, detection will require multiple ;detectors or, alternatively, a single detector with a rotating or oscillating multiple wavelength filter. Filters situated in front of each detec~or are chosen 20 ~ ~ to limit the ràdiation incident on the coIresponding detector to the emission maxima of the f luorescent label while respecdvely bloc~cing the fluorescence of the other mateIial.
Interposed between the light source 40 and an objective 62 is a dichroic ~arn splitter 64. ~ Preferably the dichroic bearn splitter 64 is ~a low-pass interference filter with a cut-off frequency chosen to be ~etween the frequencies of maximum absorpnon and 2 5 ~ ~ ~ximùm fluorescence emission of a fluorophore of interest. The dic~oic beam splitter

6 4 thus reflects high-frequency fluorescence excldng radiation from the light source 40 and ~smits thé low;frequency radiation corresponding to the fluorescence maximum of t he fl~uorophore. Depending upon ~e type of TIR element being usedj a dichroic beam splltter may not be necèssary. (See~ e. Figure 4).
30 ~ Ihe objecave 62 is selected to image the 1ight source 40 on the end face 32 of the element 24 so as to fill~ the end ~ace 32 wi~h an image of the beam shaping aperture of the source 40, the maximum angle ~of Incidence of the ray being sdected to be less than that co~sponding to the~ numer~cal ape~ e of the l'IR element~ 24. The objective 62 is ilso selected so as tQ collect substannally all o~ the r~diation~exciting ~e end face 32 over 35 ~ ~ the mlm~cal aperture of the TIR eiement 24 and to image the end face 32 on detector 50.
;As an aid in establlshing the proper posit~oning of the 'TIR element 24, the excitation :

WO 93/20240 2 ~ 3 3 ~ ~ 3 P~r/USg3/03296 source and optics 16 and detection optics 18 are preferably provided with a positioning means, such as aperture plate (not shown), dimensioned to accept the bore 30 of the sealing member 22 and dimensioned to position the end face 32 appropnately relative to the objective 62.
The detec~ion electronics 54 can be chosen from prior art direct cuITent measurements or photon counting measurements. Those ~s~illed in the art can make any combination of excitation and detection elements to achiç~e optimum detection without devia~ing or departing ~om the spirit of this inventio~, ~
The excitation source and optics 16 and detection optics 18 can be mounted in a stationary position, where a multiplicity of reaction vessels with the total internal reflection elements are 'orought into alignment with the excitation and detection optics at - periodic intervals. Those skilled in the art can design thermal cycling carousels (not shown) or ~-Y aIrays (not shown) such that the TIR elements of the respective reaction units are appropriately aligned with the optics. Alternatively, the excitation and detec~on optics can be located on a moving pla~orrn, preferably under microprocessor control9 which aligns with each individual reaction vessel kept in a sta~onary position.

E. Ot)~er Embodiments:
Figure 3 illustrates a fused lens TIR element 64. The TIR element 64 compmses a polished cylindrical rod 66, which is made of high refractive index material. At one end of the rod 66 is a semisphe~cal lens 68 that can be glued to the rod 66, or preferably molded as an integral extrusion of the rod 66. In addition, a sealing member 70 having threads 72 is provided. The sealing member 70 is preferably folmed as an extension of the semispherical lens 68. Ideally, the rod 66, lens 68 and sealing mem'oer 70 are all fonned as one piece of the same material, such as by injection molding. The threads 72 ~5 on the sealing member 70 allow the TIR element 64 to be placed in and secured to a reaction vessel (not shown). Attached to the surface of the rod 66 are coupling sites as described above.
I Figure 4 illustrates a flat or planar TIR elernent 74 in accordance with another embodiment of the present invention. The TIR element 74 has a beveled entrance surface . .
76 and exit sur~ace 78 for the excitation and emission beams respectively, thus eliminating the need for a dichroic beam-splitting rnirror. A sealing mem~xr 80 having threads 82 is provided. ~he sealing member 80 is preferably formed as an extension of the ll~ element 74. More preferably, the TIR element 74 and the sealing member 80 are ~o~med as one piece of the same material, such as by injection molding. The threads 82 on the sealin~, member 80 allow the TIR element 74 to be placed in and secured to a reaction vessel (not shown). Attached to the surface of the TIR element 74 are coupling WO 93/2~0 ~! 1 3 3 6 ~ 3 PCr/US93/03256 sites as described above.
In the embodiments of both figures 3 and 4, it is preferred to block the light from leaving the element and exciting the bulk solution via the end face. For tnis purpose, a black or highly reflective coating is used as descri'oed above. Integral TIR elements of 5 this nature may easily be constructed by injection molding techniques using transparent polymeric materials described above. The formation of these "tapered" TIR elements is described by Lackie, et al., "Instrumentation for Cylindrical Waveguide Evanescent Fluorosensors" in Biosensors with Fiberoptic Ends, Wise, et al., eds, The HumanaPress, Inc. Clifton~ NJ (1991).
Both cylindrical and planar TIR elements and the ray trace-through have been described by Muller "Spectroscopy with the Evanescent Wave in the Visible Region of the Spectrum" in Multichannel Image Detectors, Talmi, Editor, American Chemical Society Symposium ~eries #102, (1979). The use of flat TIR elements with beveled or prismatic ends has been described in Plate, et al., "Immunoassay Kinetics at Continuous 1~ Surfaces'l, in Biosensors with Fiberoptiç Ends, supra.
, In accosdance with another preferred embodiment o~ the present invention, total internal rellection ( l lK) is used to detect target nucleic acid in a reaction vessel by a degradadve process rather than a tàrget arnplification process. This reaction may be viewed as signal ampli~lcation, however, to the extent signal amplifica~ion occurs when each molecule of target can be responsible for multiple events which cause a change in signal. The apparatus used for detecting the ~rget nucleic acid is substantially the sarne as shown in ~;igures 1-4 and the same nurnerical references will made to that apparatus although the present embodiment employ an amplificahon reaction. A reactiun vessel 20, sealing member 22, and TIR element 24 are provided as described above. In the interior of the reaction vessel 20, ~le TIR element 24 is exposed to a reaction sample 36. The reaction sample 36 contains the same buffers and sample components as before.
However, the enzymaac reagents and procedure dif~er in this embodiment. A
capture initiator, ~having a por~on of nucleotide sequences which are capable ofhybridizing with the target nucleic acidg is bound tO the element 24 by any of the methods described above, preferably covalently. The label initiator, having nucleotide sequences which hybridize with an adjacent segment of target, star~s out linked to the capture inîtiator by a sclssile linlcage, such that the label is within the penetration depth. Methods of coopling molecul~s using scissile linkages are hlown in the art and are descTibed in U.S. Pat~nt Nos. 4,876,187 and 5,011~76~ (Meiogenics), which are incorpor~Lted herein by re~erence. As described ~herein, a scissile linkage is a connecting chemical structure which 30ins two nucleic acid sequences and which is capable of being selec~vely cleaved ~<'~'; '.
WO 93~20240 't~ 133 ~ 4 3 P~/US93/03256 in the presence of an appropriate enzyme and complementary target strands without cleavage of the nucleic acid sequences to which it is joined. Exarnples of scissile linkage include, but are not limited to, RNA, DNA, amino acid sequences, and carbohydrate polymers such as cellulose or starch. The reaction sample is then treated under 5 conditions sufficient to hybridize the linked initiator ~equences and target nucleic acid, if present in the reaction sample. ;~;
An agent capable of cleav~ng the scissile linkage when it is hybridized ~o target is also present in the reaction sarnple. For instance, if the scissile linkage is an RNA
sequence, an RNase is present in the reaction sample. It is within the skill in the art to 10 ~etennine empirically the types of agents needed to cleave certain scissile linkages, as well as the amount of agent to be added to the reaction sample. As the agent cleaves the scissile linkage, the fluorescing label initiator is free to dissocia~e frorn the TIR element 24 and move outside of the penetration depth. During the course of the cleavage reaction, a change (declease) in fluorescence may be detected using the total internal reflection 15 techniques described above as a measure of the presence and concelltration of target nucleic acid present in the reaction sample 36.
In accordance with yet another emb~diment of the present invention, an improYed method and apparatus f~r perforrning TIR detection of specific binding assays, including immunoassays, to detect or quanti~ate a targe~ molecule or analyte using total internal 20 reflecdon and differential temperature cycling are provided. As referenced in the "Background Descrip~on" section above, immunoassays using total internal reflection .

techniques are known in the art. Such imrnunoassays typically detect the presence of diverse target molecules of interest such as hap~ens, antigens and antibodies in reaction samples.
2~ The apparatus used for performing the immunoassay is substantially the same as shown in ~;igures 1-2 and the same numerical references will made to that apparatus although the present em~odiment need not employ a nucleic acid amplification reaction.
t A reaction vessel 2pt, a sealing member 22, and TIR element 24 are provided as discussed above. Alternatively, the integral element and sealing means shown in figurcs 3 and 4 3 0 may be used in ~hls embodiment. In the interior of the reaction vessel 2û a IIR element 24 is exposed to a reaction sample 36. ' HoweYer, th~ reacnon vessel or eell 20 of the present invention is considerably different from the TI~ vessels of the prior art. As mentioned in the "BackgroundDescnption", prior art vessels consisted of flow cells o~ capillary devices due to the need to minimlz~ dif~usion distances. By con~rast, the present reaction vessel is termed a "statlc-volumetric" cell. The modifier "sta~c" is selected because the cell is sealed or ::

WO ~3/20~40 ~! 1 3 3 6 4 3 PCI/US93/032~6 closed to other chambers; there is no flow into or out of the cell as in prior art flow cells.
The ~odifier "volumetnc" is selected because the cell encompasses a greater volume than a capillary tube. Shapes that are "volumetric" include spheres, cylinders, cubes and the like. Perhaps more importantly,"volumetric" is used to define a relationship ~etween the 5 element surface and the vessel wall that is not conducive to and even prohibits capillary nL~gration. C~il]ary TIR systems utilize the capillary migration of the fluid to flow across the element. Capillary migration is dependent on the surface tension of the fluid, the distance between the walls of the channel and the hydrophilicity of the wall surfaces For aqueous solutions using a wettable glass element and vessel9 a distance of 1.5 mm or 10 less between channel walls is essential to permit capillary action. This distance increases as the surface tension of ~he sample decreases or as the hydrophilicity of the channel walls increases. Thus, preferred volumetnc cells made of glass or similar wettable rnaterials have channel sizes of 1.7 mm or more, preferably 2.0 mm or more. Thus, the term "static-volumetric" excludes the prior art flow cells and capillary tubes.
The reaction sample 36 may comprise various specific binding reagents known in the art and preferably includes a target molecule. The target r~lolecule may be, for example, a nucleic acid, an antigen or hapten, an irnmunoglobulin, or any other protein of interest. The assay is performed using the TIR element 24 as a solid support forir.nmobilizing reac~ion sample 36 components. Those persons skilled in the art will be 20 able to determine appropriate assay configurations (e.g. sandwich or competitive) and select appropriate reaction sample 36 components (e.g. anti-analyte antibodies) which may be conjugated to a fluorophore. The prior art proYides much guidance in this regard.
The assay is performed while the reaction vessel 20 is exposed to differential tempe~an~re cycling. It will be apparent to those skilled in the art that temperatures 25 applied to the reaction vessel 20 should be selected in v~ew of the temperature sensitivity requirements of the reaction sarnple 36 components and target molecule. For instance, if the target molecule is an irnmunoglobulin, excessive temperatures, either low or high, should not be applied so. as to avoid adverse affects on the target. Nevertheless, c~cling of temperature within tolerable ranges is acceptable and within the scope of the present 30 inven~ion. Ille binding assay temperature may be con~olled manually or regulated automatically by a thermal cycler device.
Without intending to limited by any particular theory of operation, it is believed ~at temperature cycling promotes efficient diffilsion of the reaction sample to and from the surface 38 of the ~R element 24. It is aIso believed that the differential temperature 35 cycling induces convection~ currents in the lluid medium, and that the convec~ion cu~Tents enhance the diffusion of targe~ molecules in the reaction sample to the TIR element WO 93/20~40 2 1 ~ ~ 6 ~ 3 PCI/US93/03~56 surface 38, and thus enhance binding and detection of fluorescent signals. In this rnanner, a "static-volumetric" cell can be used for TIR detection of a binding assay.
It should also be realized that this temperature cycling provides for additionaldiffusion (and benefit) in nucleic acid amplification reactions such as PCR and LCR, but 5 neither is essential to the other. In other words, the nucleic acid embodiment can operale without temperature cycling and without a "static-volu~e~ric" cell, but these are both preferments for amplification. Similarly, the "stati~ ~olumetric" cell embodiment is not dependent on amplification, or even on nucleic acids for that maner, but they are preferments for this embodiment.
10 ~ The invention will now be further described by way of examples. The exarnples are intended tO ?De illustrative only; the invention is limited only by the appended claims.

EXAMPLE 1- 5y~
1~ Initiator sequences are synthesized according to standard protocols using cyanoethylphosphoramidite chesnistry and a model 380B DNA synthesizer ~Applied Biosystems, Foster Citg, CA). Various initiators are provided below in Table 3, as well as in the SEQUENCE LIST~G of the present application, where A = adenosine, C =
cytidine, G = guanosine, T = thymidine, M - aminomodifier 2TM, which introduces a prima~y amine residue (Clontech, Palo Alto, CA), and F = fluorescein (fluorescein phosphoramidite, Peninsula Laboratories, Belmont, CA). The PCR and LCR sequencesiden~fied, when used in PCR or LCR respectiYely, will amplify or capture by hyb~diza~on, portions of the Ll region of human papilloma YiI'US (HPV). These PCR
and L~R probes are disclosed in co-pending, co-owned applications Serial Nos.
07/589,948? 07/590,105 andlor 07/590,253 all filed September 28, l9gû.

:

~: : : :

:~: ` : :

: ~

}: ~
WO 93/20240 PCr/US93/03256 , Sequence Sequence . ?
_ ~ , .
1 PCR1 5'-CGTTTTCCATATTTTTTTGCAGATG 3' 2 PC R2 5'-FAATTGTACCCTAAATACCCTATATTG-3' `.
3 PCR3 s ~ - MCGTTTTCCATATTTTTTTGCAGATG-3' 4 PCR4 5'-MTTTTTTTTTTTTTTTTTTTTCGTTTTCCATATTTTTTTGCAGATG-3' capture 5'-MAAGTTGTAAGCACCGATGAATATGT-3' initiator 1 6 capture s ' -MTTTTTTTTTTTTTTTTTTTTAAGTTGTAAGCACCGATGAATATGT-3' initlator 2

7 LGR1 5'-ACATATTCATCCGTGCTTACAACT-3'

8 LCR2 s ' -TGCACGCACAAACATATATTATCAF-3'

9 LCR3 s ~ -FATGATAATATATGTTTGTGCGTGCA~3' LCR4 5'-MAAGTTGTAAGCACGGATGAATATGT-3' 11 LCR5 5'-MTTT~'TTTTTTTTTTTTTTTTAAGTTGTAAGCACGGATGAATATGT-3' 12 LCR6 s ~ -GCGGACAGGCGGAAGTTGTAAGCACGGATGAATATGT-3' 13 capture 5'-CCGCCTGTCCGCM-3' initiator 3 14 capture 5'-CCGCCTGTCCGCTTTTTTTTTTTTTTTTTTTTM-3' initiator 4 .
EXAMPLE 2 ~ Qf Initiator to S~ren~laleic Anhvdride TIR Element Chemically activated TIR elements are prepared as described in the specificauon above using styrene znaleic anhydride (commercially available frorn ARCO Chemical Company). Initiator sequence 3, 4, 5, 6, 10, 11, 13, or 14 (described in Table 3, Example 1) is separately dissolved in sodium carbonate buffer (0.1 M Na~CO3, pH 9.0) to a concentration of 16 ~M (1 x 1015 molecules/100 ~ 50 ~Ll aliquots of the initiator are then mixed with 50 ~L. of a solution of 0.02 M 1-3-e~hyl-3-(3-dime~hylaminopropyl)-carbodiimide (~DAC) and incubated ove~night at room temperature with that portion of the TIR element 24 (Figure 1) which protrudes into the reaction sarnple 36, in order to couple the aminated 5' (initiators 3, 4, 5, 6, }0 and 11) or 3' (initiator 13 and 14) end of the initiator sequence to the TIR element. Each TIR
element is washed 3-5 time$ wi~ a stream of water to remove uncoupled initiator se~uences. ~ l EXAMPIE 3 - C~i~z of Initiator to Glass TIR Element Glass elements made from commercially available glass rods are chemically derivatized utilizing 3-amino propyl triethoxysilane (Ald~ich Chemical Company) in 1%
methanol-0.001% lM hydrochloric acid with oven heating at 75~ C overnight. TIR
elements are then rinsed in 0. lM sodillm phosphate buffer pH 7.5 followed by 3-4 rinsings in distilled water and are air dried~ l~e derivauzed glass ~IR elements are then reacted with succinic anhydride for 20 - 60 minutes tO provide a linka~e site for initiator '~1336~3 `~
W093/~0240 PCr/US93/032~6 -26- i sequences. Initiator sequence 3, 4, 5, 6, 10, 11, 13, or 14 (described in Table 3, Example 1) is separately dissolved in 0.1 M sodium phosphate buffer, pH 7.5, to a concentration of 32 ~LM (2 x 1015 molecules/100 ~Ll). That portion of the TIR element 24 (Figure 1) which protludes into the reaction sample 36 is immersed in 100 ~11 of a solution of 0.02 M 1-3~ethyl-3-(3-dimethylaminopropyl)-Garbocliimide (EDAC) in phosphate buffer for 30 rr~inutes at room temperature? and 100 ~11 of initiator is then added. Incubation proceeds overnight at room tempèràture to couple the aminated 5' or 3' end of the sequence to the TIR element as in the previous example. Each TIR element is washed 3-5 times with a stream of water to remove uncoupled initiator sequences.
EXAMPLE 4 - PCR Usin~ Initiator Coupled to TIR Element Reaction units 14 ~Figure 1) are assembled comprising TIR elements 24 with PCE~3 ~described in Table 3, Example 1) covalently attached, and the reaction vessels 20.
The following reagents are added to each reaction vessel 20 to a total volume of 100 at 90C: 1 pmole PCR2; 1 unit e~nus therrnophilus DNA polymerase; 100 nmole each dATP, dCTP, dGTP, and dl'rP; and either 1 ng human placental l:)NA or a sample containing approximately 1 ng of Human Papilloma Virus DNA; all in a buffer of 100 n~ NaCl, S0 mM MgC12, pH gØ PCR proceeds essentially as described by Saiki, et al., Science, 230:1350-1354 ~1985). The reaction vessels 20 are subjected to 35 cycles of alterna~ing temperature: 1 minute at 94C, 1 minute at 65C, and 2.5 minutes at 72C. Following PC~, the reaction vessels 20 are cooled to 25C, and the fluorescence along the TIR elements is measured.
:
EXAMPLE 5 - PCR Usin~ Initiator Coupl~lement bv~lY T SPacer Reaction units are assembled and PCR is performed as described in Example 4 abo~e, except that PCR4 (described in Table 3, Example 1) is covalently attached to TIR
elements. PCR4 consists of a 3' segment identical to PCR3 that is coupled to a 5' spacer se~snent~of poly T. Following PCR, the reaction vessels are cooled to 25'C7 and the ,;
fluorescence along t~e TIR elements is measured.
EXAMPLE 6 - PCR U,sine Capture Initiaror~CouPl~ed to TIR Element -Reaction ur~its are assembled and PCR is perfoImed as described in Example 4 ;~ above, except that capture iDltiator~l (described in Table 3, Example 1) is covalently attached to TIR elements. The reaction sample is as in Example 4, except 1 pmolePCR1 is also added. ~e PCR proceeds with PCRl and PCR2 as ini~ator pairs for 35 cycles as described in Example 4. Following PCR, the reaction vessels are cooled tO

13~3 WO 93/2~240 PCr/US93/03~56 2SC, resulting in hybridization of capture initiator 1 to the fluorescein-labeled (-) strand of the amplicon. The fluorescence along the TIR elements is then measured.

EXAMPLE 7 - PCR Using Capture Initiator Coupled to TIR Element bv po~y T Spacer S Reaction units are assembled as described in Example 4 above, except that capture initiator 2 (described in Tab~e 3, Exarnple 1) is covalently attached to TIR
elements. Capture initiator 2 is identical to capture initiator I except that it is coupled to a poly T spacer segment. The reaction sample is as in Example 6, and PCR proceeds for 35 cycles as described in ~xample 4. Following PCR, the reaction vessels arecooled to 25C, resulting in hybridizanon of the 25-base HPV recognition portion of capture ini~iator 2 to the fluorescein-labeled (-) s¢and of the amplicon. The fluorescence along the TIR elements is then m~asured.

EXAMPLF g - LCR Us~ Initiators Coupled to TrR Element Reac~ion units are assembled as descnbed in Example 4 above, except that LCR4 (described in Table 3, Example 1 ) is covalenlly a~tached tO TJR elements. The reaction sample includes 1700 units Thermus therrno~hilus DNA ligase, 10 ~LM NAD, 0.1 pmole each initiator sequence LCRI. LCR2, and LCR3 in a buffer comprising SO mM N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid) (EPPS), 30 mM
Mg(:12, 0.01% bovine seIum albumin, pH 8Ø The reaction sample is assembled at 8SC. The ligase chain reaction (LC: R) proceeds essentially as described by Bac~anan and Wang [EP-A-320 30B (1988)]. The reaction vessels are subjected to 35 cycles of alte~a~ing temperature: 1 minute at 85C and l.S minute at 50~C. Following LCR, the reaction vessels are cooled to 25~C and the fluorescence along the TIR elements is measured.

EXAMPLlE 9 - LCR Usin~ Initiat~_upled to TIR Element bY pol~T Spacer Reaction units are assembled as described in Example 4 above, except tha~ LCRS
(described in Table 3, Example 1) is covalently attached io TIR elements. LCR5 consists of a 3' segment iden~ical to LCR4 that is coupled to a 5' spacer segment of poly T. The reaction sample is assembled and LCR is performed as in Example 8. Following LCR, the reaction vessels are cooled to 2S-C,~and the fluorescence along the TIR elements is measured.

: ::

.
WO 93/20~0 PCr/US93/03256 -2~-E~PI~E 10 - LCR Usin~ Ca~ture Initiator Cou~led to TIR Element Reaction units are assembled as described in Examp}e 4 above, except that capture initiator 3 (described in Table 3, Example 1) is covalently attached to TIR
elements. The reaction sample is as in Exa~ple 8 above, except 0.1 pmole LCR6 is5 also added. LCR6 consists of a 3' segment identic~-to LCR4 that is coupled to a S' segrnent complemen~y to and hybridizable ~ith capture initiator 3. LCR proceeds for 35 cycles as described in Exarnple 8. FoIlowing LCR, the reaction vessels are cooled to 25C, resulting in hybridization of the 12-base single-stranded segment of LCR6 to, the capture initiator coupled to the sur~ace of the TIR element. The fluorescence along the

10 TI~ elements is then measured.

EXAMPLE 11 - I,CR Usin~ (~aptu_Initiator,CQu~led to,llR Element bv ~olv T SpacerReaction units are assembled as described in Example 4 above, except that capture initiator 4 (described Table 3, Example 1) is covalently attached to TIR elements.
15 Capture initiator 4 is identical to capture initiator 3 except for a poly T spacer segment at the element (aminated) end. The reaction sarnple is as in Example 10. The LCR
proceeds for 35 cycles as described in Example 8. Followin~ LCR, the reaction vessels are cooled to 25~C, resulting in hybridization of the 12-base single-s~anded tail of LCR6 to the 12-base tail-complemelltary portion of capture initiator 4 coupled to the surface of 20 the l~R element. The fluorescence along the TIR elements is then measure~
While the invention has been shown and described in connection with particular prefe~red e~iments, it will be apparent that certain changes and modifications, in additlon to ~thos mentioned above, may be made by those who are skilled in the art without depar~ing from the basic fea~ures of the present invention. Accordingly9 it is the 25 intention of the Applicants to protect all vanations and modifications within the true spint and valid scope of the inven~on.

~. ~ ` ' s .

~, :

~ 21336~3 WO 93/~0~40 PCT/US93!03256 SEQUENCE LISTING

~1) GENERAL INFORMATION:
~i) APPLICANT: Abbott Laboratories (ii) TITLE OF INVENTION: METHOD AND DEVICE FOR DETECTION 3F
NUC~EIC ACID OR ANALYTE USING TOTAL INTERNAL REFLECTION
) NUMBER OF SEQUENCES: 1 ~iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Abbott Laboratories -(B) STREET: One Abbott Park Roa~
(C)-CITY: Abbott Park (D) STATE: Illinois ~E) COUNTRY: United States of America (F) ZIP: 60064-3500 ~v) COMPUTER READABLE FORM:
~A) MEDIUM TYPE: Floppy ~isk B) COMPUTER: IBM PC compatible ~C) OPERATING SYST~M: PC-DOS/MS-DOS
tD) SOFTWARE: PatentIn and WordRerfect ~vii) PRIOR APPLICATION DATA:
-~A) DOCUMENT NUMBER: 07/863,553 (B) COUNTRY: U.S.
(C) FILING DATE: 06 APRIL 1992 (viii) ATTORNEY/A~ENT INFO~MATION: :
A) NAME: Brainard, Tho~as D.
tB) RECISTRA~TIoN NUMBER: 3~,459 : (C) R~FERENCE/DOCKET NUMBER: 5158.US.01 : (ix) TELECOMMUNïCATION INFORMATION:
(A) TELEPHONE. ~708) 937-4884 B) TELEFAX: ~708) 937-2623 2)~INFORMATION:FOR SEQ ID NO~:1:
.: ::~ : (:i) SEQUENC~ CHARACTERISTICS:
(A) LENGTH: 25~base pairs TYPE: nuclei~ acid (C)~:STRANDEDNESS: qingle D~TOPOLOGY: linear ii? MOLECULE TYPE: DNA (genomic) (xi~ SEQUENCE DESCRIPTION:: SEQ ID NO:1:
: ~: : : . : :
~ CGTTT~CCAT ATTTTTTTGC AGATG; 25 , :: `:

2 133 6 ~3 P~/US93/032~6 WO g3/~240 -30- :

(2) INFORMATION FOR SEQ ID NO:2: ., (i) SEQUENCE CHARACTERISTICS: ;
~A) LENGTH: 26 base pairs~, ~B) .TYPE: nucleic acid ~.
~C) STR~NDEDNESS: single (D) TOPOI!OGY::linear (ii) MOLECULE T~PE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: misc ~eature ~B) LOCATION: 1 ~xi.) SEQUENCE DESCRIPTION: SEQ ID NO:2:
~ AATTGTACCC TA~TACCCT ATATTG : ~ 26 . ~2j INFORMATION FOR SEQ ID NO:3:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs B) TYPE: nucleic acid ~C) STRANDEDNESS: single D) TOPOLOGY: linear tii):MOLECU~E TYPE: DNA ~genomic) (ix) FEATURE: :
~ ~A~ NAME/KEY:~misc feature~
: ~ ~B) LOCATION: I
~xi) SEQUENCE~:DESCRIPTION: SEQ ID NO:3:
~ ~ :
CGTTTTCCAT ATTTTTTTGC AGATG ~ : 25 (2)~:INFORMATION FOR SEQ ID NO:4:
(i:):SEQUENCE CHARAC~ERISTICS:
(A) LENGTH::45 base pairs ~ ~
B) TYPE~: nùcleic acid : - :
C) STRANDEDNESS::single D) TOPOLOGY: lineàr ~ :
(ii) MOLECULE TYPE: DNA ~genomic) (ix)~F.E~TURE~
A) NAME/KEY: misc feature (B):LOCATION::1:
(xi) ~SEQUENCE ~DESCRIP~TION~: SEQ ID NO:4:
TTTTTTTTTT~TTTTTTTTTT CGTTTTCCAT~ATTTTTTTGC AGATG ~ 45 2~INFORM~TION:FOR 5EQ ID NO:S:
i) SEQUENCE~CHARACTERISTICS~
A) LENGTH~: 25~base pairs ; (B) TYPE:~nucleic acid C) STRAN~EDNESS: single 3 A3 6~
WO 93/2024~ PCI`~US93/03256 tD) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) ~ix) FEATURE:
~A) NAME/KEY: misc_feature ~B) LOCATION: l ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:S:

t2) INFORM~TION FOR SEQ ID NO:6:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 45 base pairs (B) TYPE: nucleic acid ~ (C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~iij MOLECULE TYPE: DNA ~genomic) ~ix) FEATURE:
tA) NAME/KEY: misc feature ~B) LOCATION: l ~xil SEQUENCE DESCRIPTION: SEQ ID NO:6:

~2) INFORMATION FOR SEQ ID NO:7:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 24 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single ~D~ TOPOLOGY: linear OLECULE TYPE: DNA ~genomic) ~xi) SEQUENCE DESCRIPTIOM: SEQ ID NO:7:

~2) INFORMATION FOR SEQ ID NO:8:
~i) SEQUENCE CHARACTERISTICS:
A) LENGTH~, 24 basie pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single ,di : (D) TOPOLOGY: linear `~
(ii) MOLECULE TYPE: DNA (genomic) (ix~ FEATURE: :
(A) NAME/KEY: misc feature : ~ (B~ LOCATION: 24 :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

: :
.
~ (2) INFORMATION FOR SEQ ID NO:9:

21336~3 W0 93/20240 ^ PCI~US93/i03256 -3~- i (i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 25 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: ~inyle ~D) TOPOLOGY: linear tii) MOLECULE TYPE: DNA (genomic) ~ix) FEATURE: :
(A) NAME~KEY: misc feature (B) LOCATION: 1 (xi) SEQUENC~ DESCRIPTION: SEQ ID NO:9:

(2) INFORMATION F~R SEQ ID NO:10: ~ :
(i) SEQUENCE CHARACTERISTICS: . .-(A) LENGTH~ ~5 base pairs (B~: TYPE nucleic acid ~: (C) STRAND DNESS: sin~le ) TOPOLOGY: linear ~;M~ MOLECULE TYPE: DNA (genomic) ;: (ix) FEATURE: :
A) UAME/KEY: misc feature : : (B) LOCATION: 1 (xi) SEQUENCE DESCRIPTION: SEQ:ID NO:10:
:: :
AAGTTGTAAG CACGGATGAA TATGT : . 25 2) INFORMATION FOR SEQ ID NO:ll: :
i) SEQUENCE CHARACTERISTICS:
A) LENGTH:~45 base pairs (B) TYPE::;nucleic:;acid ~ :
(Cj: SI'RANDEDNESS:~isingle ;~
: (D~ TOPOLOGY: linear : :~
ii) MOLECUIE~TYPE~::DNA (genomic) (ix) FEATURE~
::~ : (Aj NAME/KEY: misc feature~ . 3 B) LOC~TIdN: l :
xi)~ SEQUENCE DESCRIPTION: SEQ ID NO~
TTTTTTTTTT TTTTTTTTTT~AAGTTGTAAG CACGGATGAA TATGT~ 45 (2)~INFO~ATION~FOR~SEQ;ID~NO:~12: ~
SEQUENCE~CHARACTERISTICs: ~ j (:A~ LENGT~ 37~base~pair~
:::;S :~ (B~:::TYPE~ nucleic acid~ :
C) S~RANDEDNESS:~single~ :
(D) TOPOLOGY: linear~
MOLECULE TYPE::DNA ~genomic) i336~
. ..
. ..
WO 93/20240 PCI/U~93/032~6 i (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

~2) INFORMATION FOR SEQ ID NO:13:
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 b~se pairs ~B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: misc feature _ (B) LOCATION: 12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
~ CCGCCTGTCC GC 12 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 baqe pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY:: linear (ii) MOLECULE TYPE: DNA (genomic) (ix): FEATURE:
(A) NAME/XEY: mi c feature (B) LOCATION: 32 .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

:

`: : :

' ~ `' :

~:
:'~::

Claims (36)

WHAT IS CLAIMED IS:

1. A method of detecting amplified target nucleic acid using total internal reflection, comprising the steps of:
providing a reaction vessel having disposed therein (a) a reaction sample, (b) atotal internal reflection (TIR) element, (c) a plurality of members of initiator sequence sets and reagents for producing amplification of target nucleic acid present in the reaction sample, (d) label means which is coupled to a fluorophore, and (e) capture means for bringing said fluorophore within the penetration depth of said element, wherein at least one of said label means and said capture means is specific for said target nucleic acid;
producing an evanescent electromagnetic wave in the TIR element which penetrates into the reaction sample adjacent the element and has an associated penetration depth;
reacting the reaction sample, the initiator sequences and amplification reagentsunder conditions sufficient to amplify target nucleic acid present in the reaction sample to produce amplification products;
capturing said label means within the penetration depth as a function of the presence or amount of target nucleic acid; and detecting within the TIR element a change in fluorescence.

2. The method of claim 1 wherein a plurality of at least one member of the initiator sequence set are coupled to a specific binding member which serves also as the capture means or label means.

3. The method of claim 2 wherein said specific binding member comprises a hapten and either the capture means further comprises antihapten antibody immobilized on the element, or the label means further comprises antihapten conjugated to a fluorophore.

4. The method of claim 2 wherein said specific binding member composes a polynucleotide tail and either the capture means further comprises a complementary polynucleotide tail immobilized on the element, or the label means further comprises a complementary polynucleotide tail conjugated to a fluorophore.

5. The method of claim 1 wherein a plurality of at least one member of an initiator sequence set are coupled to the TIR element by covalent bonding.

6. The method of claim 5 wherein said plurality of at least one member of an initiator sequence set are coupled to the TIR element via a spacer molecule.

7. The method of claim 1 wherein a capture probe complementary to a portion of the amplified target is immobilized on said TIR element.

8. The method of claim 1 wherein the reaction vessel is a sealed, static-volumetric vessel.

9. the method of claim 1 wherein the reaction sample, initiator sequence sets and amplification reagents are reacted under thermal cycling conditions.

10. The method of claim 9 wherein the amplification reagents include an enzymatic agent that induces amplification, said enzymatic agent selected from thermostable DNA polymerase, thermostable DNA ligase or a combination thereof.

11. The method of claim 10 wherein the target nucleic acid present in the reaction sample is amplified by polymerase chain reaction or ligase chain reaction.

12. The method of claim 1 wherein the reaction sample, initiator sequence sets and amplification reagents are reacted under isothermal conditions.

13. The method of claim 1 wherein the step of producing an evanescent wave adjacent to the TIR element comprises directing a TIR emitted by an excitation source onto the TIR element and totally internally reflecting the beam the TIR element.

14. An apparatus for both amplifying and detecting target nucleic acid, comprising:
a sealed, static-volumetric reaction vessel adapted to contain a reaction sample and reagents for amplification a total internal reflection (TIR) element disposed in said reaction vessel such that substantial surface area of the element is in contact with said reaction sample and such that one end of the element protrudes from the vessel;
means for producing an evanescent electromagnetic wave in the TIR element which penetrates into the reaction sample adjacent the element and has an associated penetration depth;
temperature control means for reacting the reaction sample and amplification reagents under cyclic temperature conditions sufficient to amplify target nucleic acid present in the reaction sample and to capture a fluorophore capable of emitting fluorescence within the penetration depth of the element as a function of the presence or amount of target in the sample; and means for detecting in the TIR element a change in fluorescence.

15. An apparatus according to claim 14 wherein the means for producing the evanescent electromagnetic wave comprises an excitation source and optics, wherein a beam emitted from the excitation source is directed by the optics onto the protruding portion of the TIR element, the beam being totally internally reflected in the TIR element.

16. An apparatus according to claim 14 wherein the means for detecting the change in fluorescence comprises a photodetector and, optionally, optics for channeling the fluorescence from the TIR element to the photodetector.

17 . An apparatus according to claim 14 wherein the means for reacting the reaction sample and initiator sequences comprises a thermocycler device having a reaction vessel disposed therein.

18. An apparatus according to claim 14 wherein said static-volumetric reaction vessel is sealed with an integral cap/TIR element.

19. An apparatus according to claim 14 wherein said static-volumetric reaction vessel and TIR element are dimensioned such that the space between the element surface and the interior wall of the reaction vessel is too great to support capillary migration of an aqueous fluid.

20. An apparatus according to claim 19 wherein said reaction vessel and TIR
element are made of wettable materials and the distance between the element surface and the interior wall of the reaction vessel is at least about 1.7 mm.

21. An apparatus according to claim 20 wherein the distance between the element surface and the interior wall of the reaction vessel is at least about 2.0 mm.

22. A method of detecting target nucleic acid in a reaction vessel using total internal reflection, comprising the steps of:
providing a reaction vessel having disposed therein (a) a reaction sample, (b) atotal internal reflection (TIR) element, (c) a plurality of members of an initiator sequence, each sequence comprising a capture segment linked to a label segment by a scissile linkage, wherein said initiator includes a portion of nucleotide sequences which are capable of hybridizing with the target nucleic acid, and wherein said label means is coupled to a fluorophore, and (d) means for cleaving the scissile linkage when target is hybridized to the initiator;
producing an evanescent electromagnetic wave in the TIR element which penetrates into the reaction sample adjacent the element and has an associated penetration depth, and detecting in the TIR element the fluorescence resulting from the linked fluorophore present within the penetration depth;
reacting the reaction sample, initiator sequences and means for cleaving under conditions sufficient to (a) hybridize target nucleic acid present in the reaction sample to the initiator sequences, and (b), in the presence of target, cleave the scissile linkage, thereby freeing the fluorophore from the penetration depth; and detecting in the TIR element a change in fluorescence.

23. An apparatus for detecting target nucleic acid, comprising:
a reaction vessel adapted to contain (a) a reaction sample, (b) a total internalreflection (TIR) element, (c) a plurality of members of an initiator sequence, each sequence comprising a capture initiator linked to a label initiator by a scissile linkage, wherein said initiator includes a portion of nucleotide sequences which are capable of hybridizing with the target nucleic acid, and wherein said label means is coupled to a fluorophore, and (d) means for cleaving the scissile linkage when target is hybridized to the initiator;
a total internal reflection (TIR) element, the TIR element having bound thereto a plurality of initiator sequences, each of which comprises a capture initiator linked to a label initiator by a scissile linkage and wherein said label means is coupled to a fluorophore;
means for producing an evanescent electromagnetic wave in the TIR element which penetrates into the reaction sample adjacent the element and has an associated penetration depth, the fluorophore being within the penetration depth;
means for reacting the reaction sample, initiator sequences and means for cleaving under conditions sufficient to (a) hybridize target nucleic acid present in the reaction sample to the initiator sequences, and (b), in the presence of target, cleave the scissile linkage, thereby freeing the fluorophore from the penetration depth; and means for monitoring in the TIR element a change in fluorescence.

24. An improved method for detecting a target molecule in a specific binding assay using fluorescence total internal reflection, said method comprising:
performing the specific binding assay in a sealed, static-volumetric reaction vessel having a TIR element disposed therein, such that a fluorophore label means is captured within the penetration depth of the element in proportion to the presence of target molecules in the sample;
subjecting the reaction vessel to differential temperatures during the assay; and detecting in the TIR element a change in fluorescence.

25. The method of claim 24 wherein said specific binding assay is an assay for nucleic acid molecules selected from DNA and RNA.

26. The method of claim 24 wherein said specific binding assay is an assay for immunoreactive molecules selected from antigens, haptens and antibodies.

27. An improved apparatus for detecting a target molecule in a specific binding assay using fluorescence total internal reflection, said apparatus comprising:
a sealed, static-volumetric reaction vessel adapted to contain a reaction sample and reagents for a specific binding assay; and a total internal reflection (TIR) element disposed in said reaction vessel such that substantial surface area of the element is in contact with said reaction sample and such that one end of the element protrudes from the vessel;
wherein said static-volumetric reaction vessel and TIR element are dimensioned such that the space between the element surface and the interior wall of the reaction vessel is too great to support capillary migration of an aqueous fluid.

28. An apparatus according to claim 27 wherein said reaction vessel and TIR
element are made of wettable materials and the distance between the element surface and the interior wall of the reaction vessel is at least about 1.7 mm.

29. An apparatus according to claim 27, further comprising an excitation source and optics and a detector means and optics.

30. An apparatus according to claim 27, further comprising means for varying the temperature of the reaction vessel.

31. A kit for amplification and detection of a target nucleic acid, comprising:
PCR amplification reagents comprising a thermostable DNA polymerase, dATP, dCTP, dTTP, dGTP and buffer;
a reaction vessel with means for sealing the vessel from the atmosphere; and a total internal reflection (TIR) element comprising a plurality of coupling sites that allow attachment of amplified target nucleic acid.

32. The kit of claim 31, wherein said TIR element comprises a plurality of polynucleotides covalently bound thereto.

33. A kit for amplification and detection of a target nucleic acid, comprising:
LCR amplification reagents comprising a thermostable DNA ligase, NAD, and buffer;
a reaction vessel with means for sealing the vessel from the atmosphere; and a total internal reflection (TIR) element comprising a plurality of coupling sites that allow attachment of amplified target nucleic acid.

34. The kit of claim 31, wherein said TIR element comprises a plurality of polynucleotides covalently bound thereto.

35. The kit of claim 31, further comprising a thermostable DNA polymerase, dATP, dCTP, dTTP, and dGTP.

36. The method of claim 1 wherein the reaction vessel is selected from the group consisting of a flow cell, a capillary tube and a static-volumetric cell.