CA2207629A1 - Methods of immobilizing oligonucleotides to solid support materials and methods of using support bound oligonucleotides - Google Patents

Abstract

The present invention provides a method for directly immobilizing an oligonucleotide to a support material. The method comprises the steps of contacting a solution of oligonucleotides with a solid support material and drying the oligonucleotide solution to the support material. Oligonucleotides and the solid support materials to which they are immobilized can be employed as capture reagents for immobilizing nucleic acid sequences which are complementary to the immobilized oligonucleotides. Hence, the hybridization capacity of directly immobilized oligonucleotides is maintained. Also provided are methods for determining the presence or amount of nucleic acid sequences in a test sample.

Description

W O96tl9587 PCTrUS95/16627 MEI~IODS OF IMMOBILlZ~G OLIGONUCLEO l lDES TO SOLID
SUPPORT MATERIALS AND METHODS OF USING SUPPORT BOUND
OLIGONUCLEO l ll~ES

This is a col-l;"~ on-in-part application of co-pending U.S. Patent Application Serial No. 08/311,462 filed on September 22, 1994.

Field of the Invention The present invention relates to oligonucleotides. In particular, the invention relates to the immobili7~tinn of short oligonucleotides to support m~t.ori~
Back,~round of the Invention Amrlific~tion reactions such as the ligase chain reaction (LCR) which is described in European Patent Applications EP-A-320-308, the gap ligase chain reaction (GLCR) which is described in EP-A-439-182, and the polymerase chain reaction (PCR) which is described in U.S. Patents Numbered 4,683,202 and 4,683,195 are well known in the art. Such nucleic acid amplification processes are becorning useful clinical diagnostic tools to, for example, construct assays which detect infectious org~nicms in a test sample.
Amplification re~ctinn~ have also found utility in research and development fields as well as forensic fields.
Nucleic acid ~mrlification techniques typically generate copies of a target nucleic acid sequence and the presence or amount of the target sequence copies can be detected using immunological assay techniques. For example, target sequence copies can be contacted with a "capture reagent" which comprises a substantially solid support m~t~ri~l such as, for example, a suspension of microparticles coated with an oligonucleotide (variably referred to as a captureoligonucleotide) which specifically hybridizes with the target sequence copies.
In this m~nn--r, target or products of an ~mplificati~n reaction can be immobilized to a capture reagent by virtue of a target sequence copy's hyhrirli7~tion with the capture oligonucleotide. Once the target sequences are immobili~d to the capture reagent, they can easily be s~a~d from, for exarnple, extraneous reactants, by s~ali,lg the solid support from the reaction ~ Lule such as by washing OI filtration. The presence or amount of the ~mplified sequences which may be immobili~d to the capture reagent can be W 096/19587 PCTrUS95/16627 detected by contacting the captured target sequence with a "conjugate". A
conjugate can c- mpri~e a detectable moiety which is ~tt~h~l to specific binding- pair member that also specific~lly binds the ~mpli~led target sequences which are immobilized to the capture reagent. By detecting the presence of the detectable 5 moiety, the presence or amount of the target sequences can be ~e~
One hlown method of immobilizing an oligonucleotide to a support m~t~ri~l uses chPmic~l croc~linkin~ agents. Typically, cros~linking agents covalently bind a support m~t~ri~l and an oligonucleotide to forrn a linking armwhich attaches the oligonucleotide to the support m~t~.ri~l For example, U.S.
Patent No. 4,948,882 discloses compounds which can be employed to covalently link an oligonucleotide to a solid support m~tPri~l However, ch~-mi~lly cro.c~linking an oligonucleotide to a support m~t-ori~l generally is a time consuming process which requires modifications to the base pairs compri~ing the oligonucleotide.
Another method of immobilizing an oligonucleotide to a support m~teri~l which is described in Saiki, R.K., et al., Proc. Natl. Acad. Sci. USA, 86:6230-6234 tl989) involves the use of "tails". Tails are ext~-n~ions of oligonucleotides that are typically around fifteen base pairs or more in length. An oligonucleotide's tail ~ fGlc~l~ially binds solid support m~t~ri~l and, ~imil~rly to 20 a cros~link-ng agent, leaves the oligonucleotide free of the support material and available for hyhri~li7~7tion- Unfortunately, tails, which are themselves nucleic acids, sometimes illt~lrelG with the oligonucleotide's ability to specifically hybridize to a nucleic acid sequence.
As evidenced by the ar.,le-, Ir,l ~1 ioneA methods of immobilizing 25 oligonucleotides to support m~t~ri~l~, it has been accepted that relatively short oligonucleotides having between about 5 and about 50 base pairs cannot he attached directly to a solid support m~teri~l without i" ~p~ g the hyhrirli7~tion capacity of the oligonucleotide. Accordingly, known methods of ~tt~ching oligonucleotides to support m~t~-ri~l~ indirectly bind oligonucleotides to support 30 m~t-ri~l~. By indirect ~tt~c-llm~ont, the oligonucleotide itself is not bound to the support m~t-ri~l and, theoretically, is free to hybridize to another nucleic acid sequence.

wo 96/19587 PCTIUS95l16627 Sun)l~l~y of the Invention The present application describes a method for directly and non-covalently immobilizing an oligonucleotide to a support m~t~-,ri~l According to the instant method, immobili7~tion of an oligonucleotide to a support m~teri~l is 5 effected quickly and without chemi~,~l morlific~tion~ to the bases comrri~ing the oligonucleotide. I~ JU1I~I1L1Y~ the hyhritli7~tion capacity of a directly immobilized oligonucleotide is not i~ ailed. Oligonucleotides and support m~teri~ls to which they are immobilized can be employed as capture reagents for immobilizing nucleic acid sequences which are complP,m~,nt~ry to the 10 oligonucleotides bound to the support m~teri~l.
The method comprises the steps of contacting a solution of oligonucleotides with a solid support m~to,ri~l and drying the oligonucleotide solution upon the support m~t-,ri~l The oligonucleotides in solution can be in the range of between about 5 nucleotides and about 30 nucleotides in length.
15 Additionally, it has been discovered that the affinity of an oligonucleotide for a support m~teri~l can be enhanced by modifying the oligonucleotides. The method may further comprise a baking step and/or an overcoating step.
The presence or amount of the support bound oligonucleotides can be detected by contacting the solid support m~t.o,ri~l, and the oligonucleotides 20 immobilized thereon, with a conjugate and detecting a measureable signal as an in~ tinn of the presence or amount of the immobilized oligonucleotides.
According to another embodiment, the soIid support m~tt-,ri~l, and the immobili_ed oligonucleotides thereon, can be cnnt~teA with a conjugate and a measurable signal can be detected as an indic~tion of the presence or amount of 25 the conjugate.
According to yet another embodiment, support bound oligonucleotides can be contacted with a test sample snspecte~l of co..l~;..i-.g nucleic acid sequences which are compl~,m~,nt~ry to the immobilized oligonucleotides to form hybritli7~tion complexes. The hybri~1i7~tinn complexes can then be contact~d 30 with a conjugate and a measureable signal can be detect~d as an in~lic~tinn of the presence or amount of any compl~m~nt~ry nucleic acid sequences in the test s~mp]e.

W O 96/19587 PCTrUS95/16627 Brief Description of the Drawin,es Figure 1 illn~tr~tes the rrincir~es of total internal reflectance (I~).
Figure 2A, 2B and 2C are, respectively, perspective, side and cross section views of a waveguide device. Figure 2C is an enlarged cross section taken alone line C-C of Figure 2B.
Figures 3, 4A-4D, SA-5B, 6A-6C, and 7A-7B are printed representations of results obtained for assays using a capture reagent conlrri~ing oligonucleotides which were immobili_ed to a support m~teri~l as taught herein.
Details of these printed data are found in Example l ~rough Example 4.
Detailed Description of the Invention Despite previous teachings, it was surprisingly discovered that an oligonucleotide comrri~ing between about 5 nucleotides and about 50 nucleotides can be directly and non-covalently attached to a solid support m~tPri~l without imr~iring the hyhri-li7~tion capacity of the immobilized oligonucleotide. While the mP~h~ni~m by which oligonucleotides directly adhere to a solid surface is not completely nn(1P.rstood, directly attaching an oligonucleotide to a solid surface means that an oligonucleotide becomes immobilized to a suRort m~tPri~l in a manner similar to adsorption. Further, direct ~tt~chmPnt of an oligonucleotide to a solid surface does not require cros~linking agents, tails or additional nucleic acid sequences to affect the immobilization. Irnportantly, when oligonucleotides are directly attached to a solid surface as taught herein, the oligonucleotides can specifically pair or hybridize with a complclllellt~,y nucleic acid sequence.
Oligonucleotides which are immobilized as taught herein can be employed to capture or otherwise immobilize cnmrlemPnt~ry nucleic acid sequences such as, for example, 2'-deoxyribonucleic acid (DNA), ribonucleic acid ~RNA), peptide nucleic acid ~NA - as described WO 93/25706) and the ]~ke to support m~t~ori~l~ to which the oligonucleotides are immobilized. Once compl~ . y sequences hybridize to the immobilized oligonucleotides, their presence can be detected using methodologies well known in the art.
For example, a capture reagent comrn.~ing a solid support having oligonucleotides directly irnmobilized thereon can be contacted with a test s~mrle. The test sample can be any liquid suspected of cont~ining a nucleic acidsequence which can speçifi~lly hybridize with the imrnobilized oligonucleotides. The capture reagent and test sarnple can be contacted for a time W O 96/19587 PCTrUS95116627 and under conditions suitable for allowing nucleic acids in the test sample, if any, and the oligoncucleotides to hybridize and thereby form hyhri-li7ati- n complexes. The hybridization comr1exes, if any, can be cont~ct~cl with a conjugate for a time and under con-litionc sllffi~iPnt to enable the conjugate to 5 specifi~ally bind any hyhri~li7~tion comrl~xes. A signal can then be fletecte~l as an in~ ti~n of the presence or amount of any nucleic acid sequences which may have been present in the test sample.
~ nmobilized oligonucleotides as taught herein can also be employed in a "one-step" assay configuration. According to such a configuration, a test sample10 suspected of cont~ining nucleic acids which are compl~m~.nt~ry to the immobilized oligonucleotides can be contacted with a conjugate for a time and under con~ition~ suitable for allowing the conjugate to bind any nucleic acid sequences which may be present in the test sample to forrn conjugate/nucleic acid cnmpl~xe~. ~lternatively, the nucleic acids which may be present in a test 15 sample may compri~e a ~letect~hl~ moiety. Nucleic acid sequences can be labeled or conjugated with a detectable moiety through, for example, nick tran~l~tic)n whereby labeled nucleotides are incorporated into a target sequence.
Conjugate/nucleic acid complexes or nucleic acids which comprise a detectable moiety can then be contacted with the support bound oligonucleotides to form 20 conjugate/nucleic acid/oligonucleotide complexes or nucleic acid/oligonucleotide compleYes A signal can then be detected as an indication of the presence or amount of any nucleic acid sequences present in the test sample.
In a ~lcrcllcd embodiment~ a method for quickly detecting the presence of an oligonucleotide in a test sample is provided. According to this 25 embodiment, a sample which is suspected of containing oligonucleotides can beconta~teA with a support matt-rial and the oligonucleotides which may be presentin the test sample can be immobilized to the support m~teri~l as taught herein. A
conjugate can then be contacted with the immobilized oligonucleotides for a timeand under con-litionc for allowing the conjugate to bind the immobilized 30 oligonucleotides. A signal can then be detected as an indication of the presence or amount of any oligonucleotides which may have been present in the test sample.
The period for which oligonucleotides which are immobilized as taught herein are conla~;led with, for ~am~le7 a test sample, conjugate/nucleic acid 35 complex-os, or a conjugate is not important. However, it is plGr~llcd that such a wo 96/19587 Pcr/uss5/l6627 contact period be kept to a ..~ i. . -,..-- for ex~mrlP less than 30 minnt~.s, more preferably less than 15 minlltss and most preferably less than 10 minntes Those sl~lled in the art will lm(l~rct~n(l that a conjugate may comrri~e a detectable moiety att~rh~d to specific binding pair lllemb~l. Detect~hl~ moieties 5 may include any compound or conv~ntion~l detectable ch~mi~l group having a detectable and m.o~nr~ble physical or çhemi~ l property variably referred to as a signal. Such detectable groups can be, but are not intende l to be limited to, zy~ ;r~lly active groups such as en:zy-mes and enzyme substrates, prosthetic groups or coenzymes; spin labels; fluorescent molecules such as fluorescers and 10 fluorogens; chromophores and chromogens; luminescent molecnl~s such as lnminç,scers, chomiltlminescers and biohlmin~sctors; phosphorescent molecules;
specifically bindable ligands such as biotin and avidin; electroactive species;
radioisotopes; toxins; drugs; haptens; polys~cçh~ri(les; polypeptides; liposomes;
colored or fluorescent particles; colored or fluorescent microparticles; colloidal 15 particles such as s~ ninm colloid or gold colloid; and the like. Additionally, a detect~kle moiety can c-nmrTi~e, for example, a plurality of fluorophores immobilized to a polymer such as that described in co-owned and co-pending U.S. Patent Application Serial No. 091,149 filed on July 13, 1993, which is herein incorporated by reference. The detectable physical or chemical property 20 associated with a detectable moiety can be detected visually or by an external means. Specific binding member is a well known term and generally means a member of a binding pair, i.e., two dirr,lent molecules where one of the molecules through chemical or physical means specifically binds to the other molecule. In addition to antigen and antibody specific binding pairs, other 25 specific binding pairs incln(lç, but are not int~.n~lecl to be limited to, avidin and biotin, antibody and hapten, cnmpl~ , y nucleotide seqll~nces or complemt-nt~ry nucleic acid sequences such as DNA, RNA or PNA, an enzyme cofactor or sllkstr~t~ and an enzyme, a peptide sequence and an antibody specific for the sequence or an entire protein, dyes and protein hin-ler~, peptides and 30 specific protein binders (e. g., ribonllcle~e, S-peptide and ribonuclease S-protein), and the like. Furthermore, binding pairs can include members that are analogs of the original binding m~mher, for example, an analyte-analog or a binding mt~mh~r made by recombinant techniques or molecular ~ongin~Pring Thus, PNAs are specific binding members for DNA or RNA. If the binding 35 member is an immunoreactant it can be, for ~Y~mple, a monoclonal or polyclonal antibody, a rec~-mbin~nt protein or recf)mhin~nt antibody, a chim~ric antibody, a Wo 96/19587 PCT/US95116627 e(s) or fr~m-o,nt(s) of the foregoing. Detectable moieties can be attached . to specific binding pair members through any ch~,mi~l means and/or physical means that do not destroy the specific binding p~ lies of the specific binding member or the detectable properties of the cletect~ , moiety.
Preferably the method herein provided is employed to immobilize oligonucleotides to a glass surface which is then employed in a waveguide configuration such as that taught in co-owned and co-pending U.S. Patent Applic~tion Serial No. 08/311,462 filed on September 22, 1994 and entided "Light Sc~tt~,ring Optical Waveguide Method for Detecting Specific Binding Events" which is herein incorporated by reference. A waveguide device's ability to be employed in an immunoassay or hyhri~ii7~tion type format is based upon a phenomenon called total int,o,rn~l reflection (TIR) which is known in the art and is described with reference to Figure 1. TIR operates upon the principle that light 10 traveling in a denser medium 12 (i.e. having the higher refractive index, Nl) and str;king the interf~ce 14 between the denser m~dinm and a rarer m~Aillm 16 (i.e. having the lower refractive index, N2) is totally reflected within the denser m~Aillm 12 if it strikes the ;nt~ ce at an angle, ~R, greater than the critical angle, ~ C where the critical angle is defined by the equation:
~ C = arcsin (N2/N1) Under these conditions, an electromagnetic waveform known as an "evanescent wave" is generated. As shown in Figure 1, the electric field associated with thelight in the denser m-oAinm forms a st~n(ling sinusoidal wave 18 normal to the intP,rf~e. The evanescent wave penetrates into the rarer m~Ainm 16, but its energy E dissipates exponenti~lly as a function of rli~t~n~e Z from the int~ e as shown at 20. A ~ .Lr,l known as "penetration depth" (dp- shown in Figure 1 at 22) 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 int~,rf~ce. rSee, ~llth~,rl~n~l et al., J. Immunol. Meth.~ 74:253-265 (1984) ~ ,fining dp as the depth where E= (e~l) Eol. Penetration depth is calculated asfollows:
d ~/N~
P 27~{sin2~R -(N2 /Nl)2}l/2 Factors that tend to increase the penetration depth are: increasing angle of in~ nce~ ~R, closely ~ hi~g indices of refraction of the two media a.e.
N2/Nl--> l); and increasing wav~,lçn~h, ~. For ~,Y~mple, if a quartz TIR
elemP,nt (Nl = 1.46) is placed in an aqueous m~linm (N2 = 1.34), the critical W O96119S87 PCTrUS95/16627 angle, 0 C, is 66~ (= arcsin 0.9178). If 500 nm light impacts the int~rf~ce at ~R
= 70~ (i.e. greater than the critical angle) the dp is approximately 270 nm.
TIR has also been used in conjunction with light sc2tt~ring detection in a technique referred to as Scattered Total Tnt.orn~l Reflectance ("STIR"). See, e.g., U.S. Patents 4,979,821 and 5,017,009 to Schutt, et al and WO 94/00763 (Akzo N. V.). According to this technique, a beam of light is scanned across thesurface of a 1 lK el~ment at a suitable angle and the light energy is totally reflected except for the evanescent wave. Particles such as red blood cells, colloidal gold or latex specifically bound within the penetration depth will scatter the light and the scattered light is detected by a photodetection means.
Figures 2A-2C illustrate a waveguide device 30 compricing a planar waveguide element 32 and a paralIel planar plate 34. The waveguide ~-lem~nt thus has parallel surfaces 36 and 38 as well as a light-receiving edge 40.
Similarly, the plate 34 has parallel surfaces 42 and 44. The waveguide t-lem~-nt32 and the plate 34 are held together in spaced parallel fashion, such that the element surfaces 38 and the plate surface 42 define a narrow channel 46. The element and plate may be held together by any convenient means, including adhesive means 48 consisting of double stick tape disposed along the edges of the element and plate. The ch~nn~l 46 is preferably rather small so as to enablecapillary transfer of a fluid sample therethrough. For example, the height should be less than about lmrn, preferably less than about O.lm~n.
The elt-mPnt 32 should be made of an optically transparent m~t~ l such as glass, quartz, plastics such as polycarbonate, acrylic, or polystyrene. The refractive index of the waveguide must be greater than the refractive index of the sample fluid, as is known in the art for effecting total int~ l reflectance. For an aqueous sample solution, the refractive index, n, is about 1.33, so the waveguide typically has a refractive index of greater than 1.35, usually about 1.5 or more. The waveguide may be a piece of plastic or glass, for example, a standard glass microscope slide or cover slip may be used.
The plate 34 may be constructed of similar m~t~ori~l~. As seen in Figures 2A and 2B, the light lect;ivillg end 40 of the waveguide el~mçnt 32 is disposed in a narrow slit 50 of a mask 52 in order to ~ e the effects of stray light origin~ting from the light source 54. ~inimi7~tinn of stray light is also irnproved by the use of light absorbing m~t~ri~l~
Light source 54 for generating the incident light beam may be a source of electromagnetic energy, in~lntling energy in the visible, ultra-violet, and near-IR

, W O96/19587 PCTrUS95/16627 spectra. The term "light" is thus construed quite broadly and is not confin~l tothe visible range, except in cases where detection is made visually. Non-visiblewav~ ,ngth~ are detected by detectors opti~ ,ed for the particular wavelength asis well known in the art. The light may be monocl~ollla~ic or polycllLo,llaLic, collim~ted or uncnllim~t~l, pol~ri7P~ or unpolarized. Preferred light sources include lasers, light t~,mitting diodes, flash lamps, arc lamps, inC~n~escent lamps and fluorescent discharge lamps. The light source used to illnmin~te the waveguide ~ ,m~,nt can be a low wattage helium-neon laser. For a portable disposable such as that described in ex~mrle 1 below, the light source can be a small inc~n~esc-~,nt light bulb powered by a battery, such as is used in pocket fl~shlight Preferably, the light source inclll(les potentiometer means for varying the intensity of the light source. ~ltern~tively, filters and/or lenses may be employed to adjust the intensity to a suitable level.
Detection means may be employed to clet~,rmine light sC~tt~q~ring produced by a light sc~tterin~ label (LSL). As seen best in Figure 2A, a LSL may be immobilized to surface 38 of waveguide el~.m~,nt 32 via interactions between specific binding members such as, for example, that between an immobilized oligonucleotide and a cognate DNA sequence. A LSL is a molecule or a m~tt-,ri~l, often a particle2 which causes inciclent light to be scattered elastically, i.e. substant,ially without absorbing the light energy. Exemplary LSLs include colloidal metal and non-metal labels such as colloidal gold or sel~.nil-m; red blood cells; and dyed plastic particles made of latex, poly~lylene, polymethylacrylate, polycarbonate or similar m~t~,ri~l~, The size of such particulate labels ranges from lO nm to 10 ,um, ~ypically from 50 to 500 nm, and preferably from 70 to 200 nm. The larger the particle, the greater the light sc~llr,~ i"g effect, but this is true of both bound and buL~c solution particles, so background also increases with particle size. Suitable particle LSLs are available from Bangs Labol~.,lies, Inc., ~rmel, IN, USA.
Instrnmt-,nt~ti-~n and visual detection means may be employed to det~-,rmine the degree of light scatt~ring produced by a LSL. Light sc~ ,. ;"g events across the entire waveguide can be monitr)red es~nti~lly .~imnlt~neously,whether by the eye and brain of an observer or by photodetection devices including CCD ca., Ir,l i1~ which form images that are ~ligiti7PCl and processedusing colll~ul~ls.
As previously mentioned, immobilizing oligonucleotides to support m~t-o.ri~l~ according to the instant invention comprises contacting a support W O96/19S87 PCTrUS95/16627 m~t~ri~l with an oligonucleotide solution and drying the solution upon the support m~teri~l Support m~t~-ri~lc or solid supports to which oligonucleotides can be immobilized are well known in the art and include m~tt-.ri~l~ that are substantially 5 insoluble. Porous m~t-.ri~l~ can serve as solid supports and may include, for .ox~mple, paper; nylon; and cellulose as well as its derivatives such as nitrocellulose. Smooth polymeric and nonpolymeric m~teri~l~ are also suitable suRort m~t~-ri~l~ and include, but are not intended to be limited to, plastics and derivatized plastics such as, for example, polycarbonate, polystyrene, and 10 polypropylene; magnetic or non-magnetic metal; quartz and glass. Preferably, quartz, glass or nitrocellulose is employed as a support mat.-rial. Solid supports can be used in many configurations well known to those skilled in the art including, but not limited to, test tubes, microtiter wells, sheets, films, strips, beads, rnicroparticles, chips, slides, cover slips, and the l~e.
Oligonucleotides according to the invention will be understood to mean a sequence of DNA, RNA or PNA. The length of an oligonucleotide which is immobilized to a support m~t~rial is largely a matter of choice for one skilled in the art and is typically based upon the length of a complçmPnt~ry sequence of, for example, DNA, RNA, or PNA which will be captured. While the length of 20 an immobilized oligonucleotide is typically between about 5 and about 50 bases, preferably, the length of an immobilized oligonucleotide is between about 5 and about 30 bases, more typically between about 10 and about 25 bases.
Synthesis of oligonucleotides is fairly routine using automated synthesizers. If desired, automated synthesizers can produce oligonucleotides 25 which are modified with t~-rmin~l amines or other groups. A useful review of coupling chemistries is found in Goodchild, Bioconju~ate Chemistr,v. 1(3):165-187 (1990).
Modified oligonucleotides may have a greater affinity for solid supports than unmodified oligonucleotides. Methodologies for modifying an 30 oligonucleotide are well known and may include the ~ litinn of ch~.mic~l groups such as ~mines, or haptens such as fluorescein to an oligonucleotide. Such modifications do not provide for covalent linkages between a support m~t~
and an oligonucleotide, but never~eless have been found to increase the affinityof oligonucleotides for support m~t-ri~l~. Mo(1ific~tion~ are pariicularly effective 35 when made to the 3' or 5' ends of an oligonucleotide.

wo 96/19587 pcT/uss5ll6627 The oligonucleotide solution that is contacted with a solid support may comprise oligonucleotides which are in solution. The concentration of the oligonucleotides in the solution is largely a matter of choice for one skilled in the art and is typically based upon how the immobilized oligonucleotides will be 5 employed. Generally the oligonucleotide solution will have an oligonucleotide concentration of between about 1 ~M and about 1 ~mM, preferably belvv~.~ about 20 ,uM and about 250 IlM. However, as previously m-onticnecl, modified oligonucleotides have been found to have a greater affinity for support m~t~ri~l~
than unmodified oligonucleotides. Accordingly, modified oligonucleotides can 10 be employed in lower concentr~tiQn ranges than unmodified oligonucleotides.
The pH of the oligonucleotide solution may be between about 6.5 and about 8.0, preferably between about 7.0 and about 7.5. Additionally, oligonucleotide solutions are preferably saline and the sodium chloride concentration of such a snl~ltinnc can vary greatly but is typically between about 75 mM and about 2 M, preferably between about 100 mM and about 1 M, and most preferably between about 120 mM and about 500 mM.
Buffering systems may optionally be inclll~ecl in the oligonucleotide solution. Buffering systems are well known and generally comprise aqueous solutions of compounds which resist changes in a solution's hydrogen ion concentration. Examples of burr~l ulg systems include, but are not inttonrl~ to be limited to, solutions of a weak acid or base and salts thereof such as, for example, acetates, borates, phosphates, phth~l~tes, citrates, carbonates and thelike. Preferably, the buffering system comprises between about 5 mM and about 250 mM tris(~, sodium citrate, or sodium phosphate, more preferably between about 10 mM and about 200 mM tris~3), sodium citrate, or sodium phosphate and most preferably between about 10 mM and about 175 mM tris(~), sodium citrate, or sodium phosphate.
The amount of oligonucleotide solution which is applied or "spotted"
upon a solid support need be large enough only to capture snfficient c~ mrlçm~qnt~ry sequences to enable detection of, for ~ox~mrlç, a captured sequence or conjugate. This is dependent in part on the density of support m~tP.ri~l to which the capture oligonucleotide is immobilized. For ç~mrle, areasof as little as 150 ~m in tii~met~-r may be employed. Such small areas are ~,eîc;l,~d when many sites on a support m~t~.ri~l are spotted with oligonucleotide solution(s). The practical lower limit of size is about l~lm in tli~mt-.tto.r. For visual detection, areas large enough to be detçcted without m~gnific~tion are W O96119587 PCTrUS95/16627 desired; for example at least about l to about 50 mm2; up to as large as l cm2 or even larger. There is no upper size limit except as dictated by manufacturing costs and user convt~niP.nce.
Once an oligonucleotide solution is contacted with a solid support, 5 evaporation is the ~lcrcl,cd drying method and may be performed at room lelllLI. .n~lllc (about 25~C). When desired, the evaporation may be performed atan elevated temperature, so long as the temperature does not significantly inhibit the ability of the oligonucleotides to specifically hybridize with complementarysequences.
The process of immobilizing oligonucleotides to a solid support may further comprise "baking" the support m~tt-ri~l and the oligonucleotide solutionthereon. Baking may include subjecting the solid phase and oligonucleotide solution residue, to Lell~craLulcs between about 60~C and about 95~C, preferably between about 70~C and about 80~C. The baking time is not critical and preferably lasts for bGlwecl- about l5 minnt~.s and about 90 min~lte~
Baking is particularly plcrcllcd when porous support m~ten~l~ such as, for example, nitrocellulose are employed.
An overcoating step may optionally be employed in the method herein provided when porous support m~t~n~l~ are employed, but when smooth polymeric or nonpolymeric supports are employed in a waveguide format, an overcoating step is particularly plc;rrclcd. Overcoating typically comprises treating the support m~tt-ri~l so as to block non-specific interactions between the support m~t~.ri~l and comrlem.qnt~ry sequences which may be in a fluid sample.
It is ~rt;r~llcd that the overcoating or blocking m~t~-.ri~l is applied before the oligonucleotide solution has been dried upon the support m~t~-n~l Suitable blocking m~t~-.ri~lc are casein, zein, bovine serum albumin (BSA), 0.5%
sodiumdodecyl sulfate (SDS), and lX to 5X Denhardt's solution (lX
Denhardt's is 0.02% Ficoll, 0.02% polyvillyl~yllolidone and 0.2 mg/ml BSA).
~ Other blockers can include detclg~ and long-chain water soluble polymers.
Casein has been found to be a ~l~rellcd blocking m~t~ri~l and is available from Sigma Chemical, St Louis, MO. Casein belongs to a class of proteins known as "meta-soluble" proteins (see, e.g., U.S. Patent 5,120,643 to Ching, et al, incul~ulated herein by reference) which are preferably treated to render them more soluble. Such ll~ lc include acid or ~lk~linP tre~tm-~.nt and are believed to perform cleavage and/or partial hydrolysis of the intact protein.
Casein is a milk protein having a molecular weight of about 23,600 (bovine beta-wo 96119587 PCT/US95/16627 casein), but as used herein, '~casein" or "~lk~line treated" casein both refer to a partially hydroly~d ~ e that results from aLcaline l~ n~ as described in example 1 of US Patent 5,120,643. An electrophoresis gel (20%
polyacrylamide TBE) of the so-treated casein shows a n~ of fr~ment.c predomin~nt1y having molecular weight less than 15,000, as shown by a diffused band below this marker.

EXAMPLES

Example 1. DNA Hybridization Assay A. DNA Waveguide Construction DNA waveguides for the detection of human genetic mutations that cause cystic ~lbrosis were constructed from glass substrates 1 cm square.
Oligonucleotides were immobilized to the glass to provide mllltiple capture sites in the reactive surface. In particular, nine different oligonucleotides, ~iesign~te~l CAT01 through CAT09 (SEQ ID Nos. 1 - 9) were applied to the glass surface of the waveguide to form a 3 x 3 array pattern such that the CAT# corresponded to the position occupied by the same number on a standard touch-tone telephone.
DNA spots were about 2 mm in ~ mPter and about 2 mm apart. The sequence and mutation site of CAT01 through CAT09 (SEQ ID Nos. 1 - 9) are shown in Table 1.1.

TABLE 1.1 Oligo Sequence Mutation De~ign~tit n SEQ ID No. De~ign~ti-n 5' to 3 CAT01 TATcATcTTTGGTGT-NH2 ~F508WT

2 CAT02 AATATCATTGGTGTT-NH2 ~F508 4 CAT04 AGTGGAGATCAACGA-NH2 G55 lD
CAT05 AGGTcAAcGAGcAAG-NH2 R553X WT
CAT06 AGGTcAATGAGcAAG-NH2 R553X
7 CAT07 TGGAGATCAATGAGC-NH2 G551D ~ R553X
8 CAT08 TGGAGATCAACGAGC-NH2 G551D ~ R553X WT
9 CAT09 TGGAGGTCAATGAGC-NH2 G551D WT+ R553X

W O96/19587 PCTrUS95/16627 The human genetic mutations are intlic~te~ by standard notation. For example, ~F508 indicates a 3 base pair deletion at position 508 of the cystic fibrosis tr~n.~mPmhr~nP conductance regulator polypeptide (J. 7.iPlt~n~ki, et al.
Genomics 10-214-228, 1991). The "WT" in~ tes the wild type or normal sequence at this position. The DNA solutions were prepared by Synthecell (Columbia, MD) and were diluted 1:20 into PBS (phosphate burr~led saline, pH
7.4) buffer and applied to the glass surface of the waveguide using the blunt end of a drill bit approximately 1 mm in ~i~m~tPr. DNA was immobilized on a clean glass surface or to a glass surface previously coated with 0.05% casein;
10 hyhri~li7~tion results were indistinguishable. The final concentr~tinns of DNA
applied to the glass surface of the waveguide ranged from a high value of 14 ~M
for CAT02 to a low of 0.9 ~M for CAT08 and was determined by comparison to the concentration of star~ng m~t~ri~l received from Synthecell. After application, the DNA solutions were allowed to dry on the chip at room 15 temperature or, on humid days between about 35% and 80% relative humidity, in an incubator set at 50-70 ~C until dry (about 10 minutes). This procedure formed nine "spots" or hyhrifli7~tion capture sites in the 3 x3 array described above. Another glass cover slip created a channel to hold the conjugate solution.
The two cover slips were offset and held together by double-sided tape (Arcare 20 7710B, Adhesives Research Inc., Glen Rock, Penn) so as to form a channel xi~ tely 16 mm wide and a~l,lu;simately 75,um thick (the thickness of the double sided tape). The channel held ~piv~ ately 25,u1 in volume.

B. Hyhrilli7~tion To evaluate DNA waveguide performance, nine additional oligonucleotides, CAT21B through CAT29B (SEQ ID Nos. 10-18) were synthesized by Synthecell with a biotin label on the 3' end. The sequences of the test DNA oligonucleotides are listed in Table 1.2.

wo 96/19587 PCT/US95/16627 TABLE 1.2 Oligonucleotide Sequence SEQ ID No. De~ign~tinn 5~ to 3 CAT21B AcAccAAAGATGATA-biotin 11 CAT22B AACACCAATGATAI~-biotin 12 CAT23B TcGTTGAccTccAcT-biotin 13 CAT24B TcGTTGATcTccAcT-bio~n 14 CAT25B CTTGcTcGTTGAccT-biotin CAT26B CTTGCTCATTGACCT-biotin 16 CAT27B GCTCATTGATCTCCA-biotin 17 CAT28B GCTCGTTGATCTCCA-biotin 18 CAT29B GCTCATTGACCTCCA-biOtlll The oligonucleotides were clesignecl and named such that CAT21B (SEQ
ID No. lO) is comple.m~o.nt~ry to CAT01 (SE~Q ID No. 1), CAT22B (SEQ ID
No. 11) is comple.~P.-Ii1, y to CAT02 (SEQ ID No. 2), et cetera to CAT29 (SEQ
ID No. 18) which is complt;- - -r~ , y to CAT09 (SEQ ID No. 9). The concentrations varied from a high of 473 ~I for CAT25B (SEQ ID No. 14) to a low of 151 ~M for CAT27B (SEQ ID No. 16). Each of the nine DNA samples were diluted 1,ul into 1 ml of hyhritli7~ti- n buffer (1% casein, 10 mM Tris pH
7.4, 15 mM NaCl), and a dirr~lell~ one was applied to each of the nine dirr~ t DNA waveguides and incubated at room temperature (a~~ " llately 23 ~C ) for 5 minutes. The surface of the DNA waveguides were washed with PBS using a wash bottle and then stored under PBS until detection of hyhril1i7~tion.

C. DetectionofHyhri-1i77tion The waveguide was illnmin~ted with a light source compricing a 150 watt in~anclescent bulb with a ca 2 mm slit aperture. The waveguide was inserted intothe light source slit so that light was shone into the 2 mm thick light receiving edge of the waveguide (see figure 2A). The waveguide was inserted into the slit at ~ x~ ly 45~ relative to the mask.
Hybri-li7~tion of the nine dirr~lellt biotin labeled DNA's was detected in the waveguide by light that was scattered from a s~l~.ninm anti-biotin conjugate.
Collni(l~l selenillm particle as described in US Patent No. 4,954,452 to Yost, et al. having a 32 O.D. concentration, at the absorption ~~ ~,. x ;. - .~-, - . wavelength of 25 546 nm, was used to ~ ctllre the conjugate. The s~lenillm conjugate was W O96/19587 PCT~US95/16627 prepared by addition of 2.5,ul of anti-~iotin antibody (polyclonal rabbit anti-biotin antibody, 1.13 mg/ml in PBS, pH 7.4- see EP 0 160 900 B 1 to Mushahwar, et al., corresponding to US Serial No. 08/196,885 which is herein incorporated by reference) to 1 ml of the selenillm colloid, followed by addition of 30 ~l of bovine serum albumin (powder BSA dissolved in water to give a 20%
w/v solution). Fifty ~1 of the conjugate solution was applied to the surface of the DNA waveguide and light was directed into the side of the waveguide to observe binding of selPnillm to the various DNA capture sites. Positive hyhrirli7~tion was visible at many sites within 1 minute. The DNA waveguides were washed with PBS to remove excess s~lenillm conjugate, illllmin~ted to effect waveguide excited light sc~tt~ring, and imaged. This visual signal was recorded using a standard 8 bit CCD (charged coupled device) ca~era (Cohu model 4815 Cohu, Inc., San Diego, CA). A digital representation of the image was created using a frame grabber (Imaging Technology Incorporated, PC VISION plus Frame Grabber Board; Woburn, Mass) in a Compac DeskPro 386/20e (Compaq Colllpu~ Corporation, Houston, TX). The ~ligiti7e~ image data file was converted and imported into Publishers PaintBrush software (ZSoft Corp., Atlanta, Georgia) from which the image was printed on a 300 dpi resolution printer. The printed image is shown as Figure 3.
The entire pattern of DNA hyhri-li7~tion was detected using the waveguide in a single image measurement and allowed det~nnin~tion of the DNA sequence of the oligo applied to the waveguide. In the case of CAT21B
(SEQ ID No. 10) and CAT22B (SEQ ID No. 11) (first two frames of rigure 4), the hyhri-li7~tion pattern was relatively simple because there was negligible sequence homology of these oligonucleotides with DNA capture sites other than CAT01 (SEQ ID No. 1) and CAT02 (SEQ ID No. 2), respectively. In the case of CAT23B-CAT29B (SEQ ID Nos. 12-18), however, ~ignific~nt sequence homology results in a more complicated binding pattern.

Example 2. Detectin~ Oli,~onucleotides Directly Immobilized to Nitrocellulose A. Irnmobili_ation of Oligonucleotides to Nitrocellulose Synthetic oligonucleotides were haptenated using conventional methodologies and the resnlting sequences are shown below in Table 2.1. The oligonucleotides were then individually diluted (1:1) in 20X SSC buffer (3 M
sodium chloride, 342 mM sodium citrate, pH 7.0) to yield three 150,uM
solutions of each oligonucleotide.

Wo 96/19587 PCTIUSg5/16627 - TABLE 2.1 Sequence SEQ ID No. 5' 3' 19 GAAATTGG~AG~ biotin 20A~ACATGGAACATCCTTGTGGGGAC-biotin 2 1GAcTTTcGATGTTGAGATTACTTTCCC-biotin Approximately 0.3 ~1 aliquots of each oligonucleotide solution were dotted to the a~ro~ ate middle of individual 0.4 cm x 5 cm strips of 0.45 ~lm and 0.5 ~Lm nitrocellulose available from Schltqich~r & Schuell; Keene, NH.
After the oligonucleotides were applied to the nitrocellulose, the nitrocellulose strips were baked in an oven at 80~C for 20 minllte B. DetectionofImmobili~dOligonucleotides A rabbit anti-biotin s~ nillm colloid conjugate was prepared as in Example 1 except the colloid was diluted 1:1 in ~ tilled water (to yield an OD of 15 at a m;-xi~", 1 l wavelength of 546 nm) before it was added to the polyclonalantibody. The resulting conjugate was diluted 1:3 in 3% casein dissolved in tris(E~) buffered saline (TBS - 100 mM tris, 150 mM NaCl, pH 7.8). 30 ~1 of the diluted conjugate was applied to one end of each of the nitrocellulose strips which had the oligonucleotides immobilized thereon. The conjugate was allowed to migrate along the length of the nitrocellulose strips (a~lo,c i " l~tely 2 minutes) before an observation was made at which point a faint red dot was developing. After a~ploximaL~ly S mimltes, a red dot was observed on all of the nitrocellulose strips in the area where the oligonucleotides had been immobilized.

Example 3. Oligonucleotide Capture Using Oligonucleotides Directly Immobilized to Nitrocellulose A. Immobili7~tion of Oligonucleotides In this ex~mrle, oligonucleotides were immobilized to nitrocellulose and employed to capture comrlrl~ ly single str~ntled DNA sequences or double stranded DNA sequences (obtained from Genosys, Woodlands, TX; and Synthecell, Cnlllmhi~, MD). One strand of the double stranded DNA compri~ed a sequence complem~nt~ry to the immobilized oligonucleotides. The oligonucleotides which were immobilized to the nitrocellulose strips can be found in Table 3.1.

W O 96/195~7 PCTrUS95/16627 TABLE 3. 1 Sequence SEQ ID No. 5' 3' The oligonucleotides were immobilize,d to nitrocellulose by dotting 150 5 ~M solutions of the oligonucleotides OlltO a~luxi,.l~tely 0.4 cm x 5 cm strips of S ~m nitrocellulose (Schleicher & Schuell). The immobilization procedure was the same as the procedure set forth above in Example 2 except that after the oligonucleotide solutions were applied to the nitrocellulose strips, the strips were baked at 75~C.
B. Hyhri~li7~tion of Lrnrnobilized OIigonucleotides and Their Cognates Initially, the cognate oligonucleotides and the double stranded sequences cnmrricing a comrlemPnt~ry oligonucleotide (both of which will hereinafter be referred to as running oligonucleotides) were at a concentr~tinn of between 100 15 ,uM and 500 ,uM. The running oligonucleotides were diluted in 1% casein dissolved in 10 mM tris, 15 mM NaCl, pH 7.4. The sequences of the running oligonucleotides can be found in table 3.2.

TABLE 3.2 SEQ ~D No. Sequence 23 5 - ACACCAAAGATGATA-fluorescein - 3 24 5 - TATCATCTTTGGTGT-nuul~sceill - 3 5 -TATCATCTTTGGTGT-fluorescein-3 26 5'-biotin-ACACCAAAGATGATA-3 Hyhritli7~tion was achieved by applying 30 ,ul of the running oligonucleotides to one end of the n*ocellulose strips and allowing the oligonucleotides to migrate past the region co~ g the imrnobilized oligonucleotides (a~plu~ a~ly 5 minutes). Detecting hyhri~li7~tiQn of the 25 running oligonucleotides to the irnrnobilized oligonucleotides was accomplich~d in one of two ways. One method included placing the strips under U.V. light and observing the situs of the imrnobilized oligonucleotides for a fluolt;scen~

CA 02207629 l997-06-l2 WO 96119~87 PCTnUS95/16627 signal. The other method inrlll-led contacting the ends of the nitrocellulose strips - with a sel~nillm colloid conjugate mixed with the running oligonucleotides, allowing the conjugate to migrate past the situs of the immobilized oligonucleotides, and observing the situs of the immobilized oligonucleotides for 5 a visible signal. A signal in the region of the immobilized oligonucleotides in~lic~te~l the hyhrilli7~tion of the running oligonucleotides and therefore thepresence of the immobilized oligonucleotides in their original position. The selenillm colloid conjugates comprised selenillm colloid and an antibody specific for the label ~tt~ch~d to the running oligonucleotide. Conjugates were prepared 10 as above in Example 2 using anti-fluorescein antibody and anti-biotin antibody.
Figure 4 is a photograph of nitrocellulose strips under a U.V. lamp after SEQ ID No. 23 migrated past the situs of immobilized SEQ ID No. 1. Figure 4(a) illustrates the fluorescent signal generated at the situs of the irnmobilized oligonucleotides when a 100 nM concentration of SEQ ID No. 23 was employed 15 as the running oligonucleotide. Figure 4(b) illustrates the fluorescent signal generated at the situs of the immobilized oligonucleotides when a 1 ~I
concentration of SEQ ID No. 23 was employed as the running oligonucleotide.
Figure 4(c) illustrates the fluorescent signal gen~r~tecl at the situs of the immobili_ed oligonucleotides when a 1.6 ~M concentration of SEQ ID No. 23 20 was employed as the running oligonucleotide. Figure 4(d) illustrates the fluorescent signal ge~ Ir,l ~l(ecl at the situs of the immobilized oligonucleotides when a 3.3 ~uM concentration of SEQ ID No. 23 was employed as the running oligonucleotide. As illustrated by the signals observed in Figures 4(a)-(d), therunning oligonucleotides hyhri-li7Prl to the immobilized oligonucleotides in the25 region where they were originally applied.
Figure 5 illustrates the results obtained from another one step format where SEQ ID No. 22 was immobili~d to nitrocellulose and SEQ ID No. 24 (single stranded DNA), at a 20 ~M concentration, and SEQ ID No. 25 (double str~n-led DNA), at a 20 ,uM concentration, were used as the running 30 oligonucleotides at a 1 :30 dilution in the nmning buffer (1 % casein dissolved in 10 mM tris, 15 mM NaCl, pH 7.4). A selenillm colloid anti-fluorescein conjugate (as prepared in Example 2) was employed to visually detect hyhTi~li7Ation Figure 5(a) shows the results after SEQ ID No. 24 and the sel~ni-lm colloid conjugate had migrated past the situs where SEQ ID No. 22 had 35 been immobilized. Similarly, Figure 5(b) shows the results after SEQ ID No.
25 and the sçleni~lm colloid conjugate had migrAted past the situs where SEQ ID

W O96/19S87 PCTrUS95/16627 No. 22 had been immobilized. As illustrated by Figures 5(a) and 5(b), hyhri-li7~tion was detected for the single stranded DNA (SEQ ID No. 22) but no hyhritli7~tion was d~tected for the double stranded DNA (SEQ ID No. 25).
Figure 6 illnstr~tes the results obtained when SEQ ID No. 1 was 5 immobilized to three nitrocellulose strips and various con~çn~T~tinnC of SEQ ID
No. 26 were employed as a running oligonucleotide. Figure 6(a) illn~tr~tes the results obtained when the running oligonucleotide was applied to the end of a nitrocellulose strip at a lO0 nM concentration. Figure 6(b) illn~tr~tes the results obtained when the running oligonucleotide was applied to the end of a 10 nitrocellulose strip at a l nM concentration. Figure 6(c) illustrates the results obtained when the running oligonucleotide was applied to the end of a nitrocellulose strip at a 0.1 nM concellh~tion. Hyhrirli7~tion of the running oligonucleotide to the immobilized oligonucleotide was detected visually, as above, using a selenium colloid anti-biotin conjugate (as prepared in Example 2).
15 As shown by Figures 6(a)-6(c), hyhri~li7~tion occurred on all three strips as indicated by the signals observed in the regions where SEQ ID No. l was initia~ly immobilized.

Example 4. Oli~onucleotide Capture Usin,~ DNA & PNA Directly Immobilized to Glass A. Directly Immobilizing Oligonucleotides and PNAs to Glass Oligonucleotides were immobilized to 22 mm x 22 mm glass cover slips from Corning and employed to capture a cognate oligonucleotide. SEQ ID No.
27 was the DNA oligonucleotide immobilized to the glass cover slip and SEQ ID
25 No. 28 was the PNA oligonucleotide immobilized to the glass cover slip. SEQ
ID No. 27 was synthesi7eA using convenitonal ~lltom~ted techniques and is listed below in Table 4.1. SEQ ID No. 28 was purchased from Millipore (Bedford, MA) and is listed below in Table 4.2.

TABLE 4.1 Sequence SEQ ID No. 5' 3 27 . ~ GAG~l~l~l~iAGTGA

W O 96/19587 PCTnUS9~/16627 TABLE 4.2 Sequence SEQ ID No. C-lr~N-tPrminlls 28 nuu-~,ce~ GAGGTTGGTGAGTGA-NH2 Two dilutions of the oligonucleotide sequences were prepared. The PNA was diluted in phosphate buffered saline (PBS) to yield solutions c~ nt~ining PNA concentrations of 44 ,uM and 11 ~lM. The DNA was diluted in PBS to yield solutions con~ il-g oligonucleotide concentrations of 37 IlM and 14 ~M. 1 111 aliquots of each dilution were then dispensed onto glass cover slips. The PNA and DNA solutions were allowed to dry onto the cover slip at room temperature before the cover slip was baked at 60~C for 20 minntes. After the cover slip was baked, it was cooled to room temperature and overcoated with a 0.05% solution of ~lk~lin~ treated casein dissolved in HPLC water. The overcoating lasted for one minute and excess casein was rinsed from the cover slip with HPLC water. Rçsi~ l liquid on the cover slip was dried with forced air.

B. Production of an Optical Waveguide Using double shck tape, a second 22 mm x 22 mm glass cover slip was secured (slightly off center) to the cover slips which had been spotted with theoligonucleotide sequences. The channel formed between the two cover slips was approximately 0.75 ~m deep and held ap~lo~ ately 25 ~11 of a liquid reagent. Two waveguides were produced in this manner.

C. Hyhri~i7~tion and Detection A DNA sequence (Genosys) which was complementary to SEQ ID No.
27 (DNA) and SEQ ID No. 28 (PNA) was employed as a sample oligonucleotide. The sample oligonucleotide is de.cign~te~l SEQ ID No. 29 and is listed below in Table 4.3.

TABLE 4.3 Sequence SEQ ID No. 5' 3 29 biotin-TCACTCACCAACCTC

WO96tl95~7 PCTrUS95/16627 Separate dilutions of SEQ ID No. 29 were made in buffers containing dirr~;lcl~ concentrations of sodium phosphate. SEQ ID No. 29 was diluted to 170 nM in 1.5 mM phosphate buffer and 150 mM phosphate buffer. An aliquot, large enough to fill the waveguide's çh~nntel, of each dilution was then 5 dispensed into the two waveguides produced above. The waveguides were then incubated at room ~Clll~)Cl~l,UlC for S minutes in a hnmi(lity chamber at 100%
humidity. After the incubation, the liquid in the waveguide was displaced with an an~*-bio*n selenium colloid conjugate which was prepared as above in Example 1 then the conjugate was diluted 1:1 with 10% ~lk~line treated casein in10 dis*lled water. Tmme~i~tely upon displacement, the waveguide was inserted into a 150 watt light source as in Example 1.
Figure 7 illustrates the results obtained when the waveguide was inserted into the light source. Figure 7(a) is a legend showing the dotting pattem used for the immobilization of the various concentr~*t ns of SEQ ID No. 27 and SEQ
ID No. 28. Figure 7(b) shows the waveguide results for SEQ ID No. 29 when diluted in 1.5 mM phosphate buffer. As shown by Figure 7(b), the signal was the greatest in the area where 44 ~M PNA was dotted. Figure 7(c) shows the waveguide results for SEQ ID No. 29 when diluted in 150 mM phosphate buffer. As shown by Figure 7(c), the signal was again the greatest in the area 20 dotted with the 44 ~lM PNA but the binding affinity for the DNA spots was greater than in the 1.5 mM dilutions.

The above examples describe several specific embodiments of the invention but the inven*on is not restricted to these specific ex~mples Rather, 25 the inven*on to be protected is defined by the appended claims.

CA 02207629 l997-06-l2 SEQUENCE LISTING
- (1) GENERAL INFORMATION:
( i ) APPLICANT: Tsung-Hui K. Jou Joanell V. Hoijer (ii) TITLE OF INVENTION: METHODS OF IMMOBILIZING OLIGONUCL~:Ol~l~S
TO SOLID SUPPORT MATERIALS AND METHODS
OF USING SUPPORT BOUND OLIGONUCLEOTIDES
(iii) NUMBER OF SEQUENCES: 29 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Abbott Laboratories (B) STREET: 100 Abbott Park Road (C) CITY: Abbott Park (D) STATE: Illinois (E) COUNTRY: USA
(F) ZIP: 60064-3500 (v) COMPUTER READABLE FORM:
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(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Paul D. Yasger (B) REGISTRATION NUMBER: 37,477 (C) DOCKET NUMBER: 5636.US.01 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 708/937-4884 (B) TELEFAX: 708/938-2623 (C) TELEX:

(2) INFORMATION FOR SEQ ID NO:1:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(2) INFORMATION FOR SEQ ID NO:2:
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CA 02207629 l997-06-l2 W O 96/19587 PCT~US95116627 (A~ LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(2) INFORMATION FOR SEQ ID NO:5:
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(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA

CA 02207629 l997-06-l2 WO 96/195~7 PCI/US95/16627 (ix) FEATURE:
(A) NAME/KEY: 3' amine (B) LOCATION: 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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50 (2) INFORMATION FOR SEQ ID NO:10:
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(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
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(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) sTR~NnRnN~cs single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(i) SEQUENCE CHARACTERISTICS-(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) sTRA~nRn~R~s: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
(A) NAME/ ~ Y: 3' biotin (B) LOCATION: 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (c) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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30 ( 2) INFORMATION FOR SEQ ID NO:17:
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(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
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(2) INFORMATION FOR SEQ ID NO:18:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - 50 (ii) MOLECULE TYPE: synthetic DNA
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(A) NAME/KEY: 3' biotin (B) LOCATION: 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

(2) INFORMATION FOR SEQ ID NO:19:

W O 96/19587 PCTrUS95tl6627 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs '(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
(A) NAME/KEY: 3' biotin (B) LOCATION: 26 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:

(2) INFORMATION FOR SEQ ID NO:20:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
(A) NAME/KEY: 3' biotin (B) LOCATION: 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
A~ACATGGAA CATCCTTGTG GGGAC 25 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
( ix ) FEATURE:
(A) NAME/KEY: 3' biotin (B) LOCATION: 27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D). TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID No : 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid CA 02207629 l997-06-l2 W O96/19587 PCTnUS95/16627 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
(A) NAME/KEY: 3' fluorescein (B) LOCATION: 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid ( c ) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
(A) NAME/KEY: 3' fluorescein (B) LOCATION: 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

25 (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
(A) NAME/KEY: 3' fluorescein (B) LOCATION: 15 (xi) SEQUENCE DESCRIPTION: SEQ ID No:25:

(2) INFORMATION FOR SEQ ID NO:26:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
~(A) NAME/KEY: 5' biotin (B) LOCATION: 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

(2) INFORMATION FOR SEQ ID No:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

WO96/19587 PCTrUS95/16627 (ix) FEATURE:
(A) NAME/KEY: 5' carbazole (B) LOCATION: l (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GAG~~ ~lG AGTGA l5 (2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l5 base pairs (B) TYPE: peptide nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other (PNA) ( ix) FEATURE:
(A) NAME/KEY: peptide bond backbone (B) LOCATION: 1-15 (ix) FEATURE:
(A) NAME/KEY: C-terminus fluorescein ( B) LOCATION: l (ix) FEATURE:
(A) NAME/KEY: N-terminus amino (B) LOCATION: l5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
GAGGTTGGTG AGTGA l5 (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) ToPOLOGY: linear (ii) MOLECULE TYPE: DNA
( ix ) FEATURE:
(A) NAME/KEY: 5' biotin (B) LOCATION: l (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
40 TCACTCACCA ACCTC l5

Claims (10)

We claim:

1. A method of non-covalently immobilizing an oligonucleotide to a support material comprising the steps of:
(a) contacting an oligonucleotide solution with a solid support material wherein said oligonucleotides comprise between 5 nucleotides and 30 nucleotides; and (b) drying said solution upon said solid support material.

2. The method of claim 1 wherein said solution has an oligonucleotide concentration of between 1 µM and about 1 mM

3. The method of claim 1 wherein said solution further comprises a pH between 6.5 and 8.0, between 75 mM and 2 M sodium chloride, and a buffering system.

4. The method of claim 3 wherein said buffering system comprises between 10 mM and 250 mM tris R, sodium citrate or sodium phosphate.

5. The method of claim 1 wherein after said drying, said method further comprises baking said solid support material at a temperature between 60°C and 95°C.

6. The method of claim 1 wherein said oligonucleotides further comprise an amine group at the a 5' end or a 3' end.

7. The method of claim 1 wherein said oligonucleotides are peptide nucleic acids.

8. The method of claim 1 wherein after step (b), the method further comprises (i) contacting said solid support material with a conjugate;
and (ii) detecting a measurable signal as an indication of the presence or amount of said oligonucleotides.

9. The method of claim 1 wherein after step (b), the method further comprises:
(i) contacting said solid support material with a conjugate;
and (ii) detecting a measurable signal as an indication of the presence or amount of said conjugate.

10. The method of claim 1 wherein after step (b) the method further comprises:

(i) contacting said solid support with a test sample suspected of containing nucleic acid sequences which are complementary to said oligonucleotides to form hybridization complexes;
(ii) contacting said complexes with a conjugate; and (iii) detecting a measureable signal as an indication of the presence or amount of said nucleic acid sequences.