Note: Descriptions are shown in the official language in which they were submitted.
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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
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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.
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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.
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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
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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
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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
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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
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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
,
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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
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-10-
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.
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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
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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-
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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
3 CAT03 AGTGGAGGTCAACGA-NH2 G551D WT
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
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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.
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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
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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.
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- 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.
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TABLE 3. 1
Sequence
SEQ ID No. 5' 3'
TATCATCTTTGGTGT-NH2
22 ACACCAAAGATGATA
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
3 -ATAGTAGAAACCACA-5
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~
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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
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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
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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
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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.
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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:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: Macintosh
( c ) OPERATING SYSTEM: System 7Ø1
(D) SOFTWARE: MS Word
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(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
(ix) FEATURE:
(A) NAME/KEY: 3' amine
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
TATCATCTTT GGTGT 15
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
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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:
(A) NAME/KEY: 3' amine
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
AATATCATTG GTGTT 15
(2) INFORMATION FOR SEQ ID NO:3:
(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' amine
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AGTGGAGGTC AACGA 15
(2) INFORMATION FOR SEQ ID NO:4:
(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' amine
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
AGTGGAGATC AACGA 15
(2) INFORMATION FOR SEQ ID NO:5:
(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' amine
(B) LOCATION: 15.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
AGGTCAACGA GCAAG 15
(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
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-25 -
(ix) FEATURE:
(A) NAME/KEY: 3' amine
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
AGGTCAATGA GCAAG 15
(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
( ix) FEATURE:
(A) NAME/KEY: 3' amine
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
20 TGGAGATCAA TGAGC 15
(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
(ix) FEATURE:
(A) NAME/KEY: 3' amine
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
TGGAGATCAA CGAGC 15
(2) INFORMATION FOR SEQ ID NO:9:
(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' amine
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TGGAGGTCAA TGAGC 15
50 (2) INFORMATION FOR SEQ ID NO:10:
(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' biotin
(B) LOCATION: 15
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
ACACCAAAGA TGATA 15
5 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) sTR~NnRnN~cs single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(ix) FEATURE:
(A) NAME/KEY: 3' biotin
(B) LOCATION: 15
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AACACCAATG ATATT 15
(2) INFORMATION FOR SEQ ID NO:12:
(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' biotin
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
TCGTTGACCT CCACT 15
(2) INFORMATION FOR SEQ ID NO:13:
(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/ ~ Y: 3' biotin
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
45 TCGTTGATCT CCACT 15
(2) INFORMATION FOR SEQ ID NO:14:
(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:
CTTGCTCGTT GACCT 15
-
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(2) INFORMATION FOR SEQ ID NO:15:
(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' biotin
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CTTGCTCATT GACCT 15
(2) INFORMATION FOR SEQ ID NO:16:
(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' biotin
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GCTCATTGAT CTCCA 15
30 ( 2) INFORMATION FOR SEQ ID NO:17:
(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' biotin
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GCTCGTTGAT CTCCA 15
(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
(ix) FEATURE:
(A) NAME/KEY: 3' biotin
(B) LOCATION: 15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GCTCATTGAC CTCCA 15
(2) INFORMATION FOR SEQ ID NO:19:
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-28-
(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:
GAAATTGGCT CTTTAGCTTG TGTTTC 26
(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:
40 GACTTTCGAT GTTGAGATTA CTTTCCC 27
(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:
ACACCAAAGA TGATA 15
(2) INFORMATION FOR SEQ ID No : 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
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-29-
(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:
ACACCAAAGA TGATA 15
(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:
TATCATCTTT GGTGT 15
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:
TATCATCTTT GGTGT 15
(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:
ACACCAAAGA TGATA 15
(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
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-30-
(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
