CA1323552C

CA1323552C - Assay for polynucleotides employing oligonucleotides to eliminate undesirable cross reactions

Info

Publication number: CA1323552C
Application number: CA000573409A
Authority: CA
Inventors: Lyle John Arnold Jr.
Original assignee: Gen Probe Inc
Current assignee: Gen Probe Inc
Priority date: 1987-07-31
Filing date: 1988-07-29
Publication date: 1993-10-26
Anticipated expiration: 2010-10-26
Also published as: EP0304184B1; KR960002234B1; ES2063038T3; DK153589D0; JP2783568B2; AU2266588A; AU623090B2; KR890701765A; US5434047A; DK153589A; PT88144A; DE3851810D1; FI891524A; NO891366L; EP0304184A1; FI891524A0; JPH02500804A; DE3851810T2; WO1989001049A1; ATE112804T1

Abstract

ABSTRACT
In an assay for a target nucleotide sequence employing hybridization with a target probe nucleotide sequence, cross reaction between the target probe and a non-target nucleotide sequence, capable of hybridizing with the target probe, is prevented or diminished by introducing, at some point during the hybridization, a non-target probe which hybridizes preferentially with the non-target nucleotide sequence. The melting temperature for the non-target probe/non-target nucleotide sequence hybrid is preferably greater than that of the target probe/non-target sequence hybrid. The hybridization can be carried out under isothermal conditions or preferentially with gradual cooling.

Description

1323~2 179/260 S P E C I F I C A T I O N
_ ASSAY FOR POLYNUCLEOTIDES EMPLOYING OLIGONUCLEOTIDES
TO ELIMINATE UNDESIRA~LE CROSS REACTIONS

Field of the Invention The present invention relates to an assay for a desired nucleotide sequence (the "target nucleotide sequence") which may be presen~ together with other nucleotide sequences (the "non-target nucleotide sequences"), the latter being capable of interfering in the assay for the former to result in potentially false qualitative or quantitative results.

Technologv review It has long been recognized that unique or misexpressed nucleotide sequences in a polynucleotide can be detected by hybridization with a nucleotide multi~er probe. This technique permits the early detection of infectiou~ organisms such as bacteria, virùses, etc.; genetic diseases such as sickle cell anemia; and various cancers. The probe (referred to as the "target proben) is selected with a nucleotide sequence complementary (usually exactly complementary) to the nucleotide sequence which is desired to be detected (the "target nucleotide sequence"). Following hybridization of the probe with the target polynucleotide, any target probe/polynucleotide hybrids which have formed are typically separated from unhybridized probe. The amount of probe in either of the two separated mediums is then tested to provide a qualitative or quantitative measurement of the amount of target originally present.

_ 1323~2 ~ owever, in many assays there may be one or more non-target polynucleotides present which have a nucleotide sequence closely related to that of the target sequence (for example differing in only 1 to 5 nucleotides). In such cases the non-target polynucleotide may then interfere with the assay, by hybridizing with at least some of the target probe, to produce false qualitative or quantitative results. This problem is exacerbated where the probe sequence is selected to permit assaying ~f various species of organisms within a particular genus, each species of which contains the target nucleotide sequence or one very closely related thereto (e.g., differing by only one nucleotide).
A solution to the foregoing type of interference has been suggested, for example by Wallace et al., Proc. Natl. Acad.
Sci. USA, 80, 278-282 (1983) where the precise control of stringency is used to detect single nucleotide differences.
Using oligonucleotide probes which were only 19 nucleotides in length and precisely controlling stringency Wallace et al. was able to diQcriminate sickle anemia associated hemoglobin DNA and normal DNA bound to nitrocellulose which differed by only a single point mutation. Such a method takes advantage of the fact that the target probe/target polynucleotide hybrid will have a highe~ melting temperature than the target probe/non-target polynucleotide hybrid, since the target probe is intentionally selected with a nucleotide sequence more closely complementary to that of the target nucleotide sequence, than to any related nucleotide sequence of any non-target polynucleotide which might be present. In these types of assays then, polynucleotides bound to nitrocellulose are hybridized with polynucleotide probes at a controlled temperature which ideally is between the respective 1~235~2 melting temperatures of the probe/target polynucleotide hybrid and the probe/non-target polynucleotide. However, the foregoing meiting temperatures may differ by only about 2-3C.
Thus, a serious difficulty with the foregoing method is that it requires stringent control of temperature, and even then either typically permits a large amount of interference from the non-target polynucleotide (i.e., relatively large amounts of probe are hybridized with non-target polynucleotide), or results in low ex~ents of hybridization between the target probe and target polynucleotide, thereby potentially leading to false qualitative results. Furthermore, for guantitative results an even higher degree of temperature control is required, since close to the same temperature must be maintained during hybridizations in both assays to establish a standard curve, and an assay of an unknown sample.

Definitions As used in this disclosure and claims, the following terms are defined as:
nucleotide: a subunit of a nucleic acid consisting of a phosphate group, a 5 carbon sugar and a nitrogen containing base. In RNA the 5 carbon sugar is ribose.
In DNA, it is 2-deoxyribose.
nucleotide multimer: a chain of nucleotides linked by . . . _ phosphodiester bonds or analog thereof.
oligonucleotide: a nucleotide multimer generally about 10 to about 100 nucleotides, but which may be 200 or more nucleotides in length. They are usually synthesized from nucleotide monomers or obtained by enzymatic means.

132~2 PolYnucleotide: a nucleotide multimer generally more than about 100 nucleotides in length.
tar~et nucleotide sequence: is a nucleotide sequence for which an assay is being performed. It may cover a group of closely related nucleotide sequences (e.g., differing by only one nucleotide such as various organisms within a genus).
non-target nucleotide seauence: is one or more nucleotide sequences which may interfere with an assay for the target nucleotide sequence using a selected probe because of a high degree of sequence homology with the target nucleotide sequence. Generally, but not necessarily, the target and non-target nucleotide sequences are on distinct nucleotide multimers. The non-target probe may be complementary with only a portion of the non-target sequence and may overlap a portion of both the non-target sequence and adjacent nucleotides. It i9 only necessry that the non-target probe hybridize with enough of the non-target sequence to prevent cross reaction with the target probe.
nucleotide multimer Probe: a probe which is itself a nucleotide multimer (u~ually, but not necessarily, an oligonucleotide), selected to hybridize preferentially with a target, or non-target, nucleotide mul~imer.
melting temPerature: with reference to a nucleotide multimer hybrid, is that temperature at which one half of that hybrid is denatured.

13235~2 60724-1829 Summarv of the Invention The present invention then, provides a means in an assay for a target nucleotide sequence using a selected probe (the "target probe"), by which interference from one or more non-target nucleotide sequences can be reduced. This is accomplished by providing one or more other probes (the "non-target probe") which can hybridize with at least a portion of the non-target sequences which may be present, so as to reduce the amount of the non-target sequences which might otherwise hybridize with the target probe.
The invention provides an assay for a target nucleotide sequence in a test sample which may contain a closely related non-target nucleotide sequence or sequences, comprising:
(a) contacting any test sample with a target probe and at least one non-target probe under conditions to permit hybridization between the target probe and said target nucleotide sequence while minimizing any undesired hybridization between the target probe and said non-target nucleotide sequence, any said latter undesired hybridization being reduced by the presence of the non-target probe which is capable of hybridizing with any non-target nucleotide sequence present in at least one of the assay steps so as to increase the target probe/target nucleotide sequence to target probe/non-target nucleotide sequence hybridization ratios wherein the hybridization conditions are established with all the nucleotides present in a liquid medium, absent any solid phase support; and, (b) assaying for hybridization of said target probe to any test sample nucleotide sequence.

Bl 5 13235~2 60724-1829 The invention also provides an assay in a liquid medium for a target nucleotide sequence in a test sample which may contain a closely related non-target nucleotide sequence comprising:
(a) contacting any test sample in a liquid medium with a target probe and at least one non-targst probe under conditions to permit hybridization between the target probe and said target nucleotide sequence while minimizing any undesired hybridization between the target probe and said non-target nucleotide sequence any said latter undesired hybridization being reduced by the presence of the non-target probe which is capable of hybridizing with any non-target nucleotide sequence present in at least one of the assay steps so as to increase the target probe/target nucleotide sequence to target probe/non-target nucleotide sequence hybridization ratios wherein the hybridizing conditions are established with all the nucleotide present in the liquid medium, absent any solid phase support on which the liquid medium is absorbed; and, ~b) assaying for hybridization of said target probe to any test sample nucleotide sequence.
The invention also pro~ides a kit useful for assaying a target multimer in a test sample which may also contain a non-target multimer, comprising.
(a) a labelled, nucleotide multimer target probe selected such that under hybridizing conditions, it can form a target --~
probe/target sequence hybrid, but which can also incidentally form a target probe/non-target sequence hybrid;
(b) an unlabelled, nucleotide multimer non-target probe, distinct from the target probe, selected such that under hybridizing conditions, it can form a non-target probe/non-target multimer hybrid so as to reduce the amount of target 5a ,.~

132~552 probe/non-target multimer hybrid which may form absent the non-target probe, while not unduly reducing the amount of target probe/target multimer hybrid which can form wherein the molar ratio of non-target probe to target probe is no greater than 9: 1.
Preferably then, the present invention provides an assay, and a kit useful in performing such an assay, for detecting a target polynucleotide sequence which may also contain a non-target polynucleotide sequence. The medium is contacted with distinct, target and non-target, nucleotide multimer probes under hybridizing conditions. This allows the target probe to hybridize with the target present (i.e., the target that may be present). However, since the target and non-target multimers are closely related, the probe can also incidentally hybridize with non-target polynucleotide sequences present. The non-target probe or probes are selected so that under such hybridizlng conditions, it will hybridize with non-target polynucleotide sequence present, so as to reduce the extent of any target probe/non-target hybridization which may occur absent the non-target probe, while not unduly reducing any target probe/target hybridization (i.e., not reducing it to such an extent as to make the assay unworkable in a practical situation).
This invention can be utilized with any of the standard hybridization methods. Generally this employs formation of the target probe/target hybrid on a surface or in solution in a 5b .
.. i ~ .
. .

1323~2 179/260 manner so as to permit detection of the target probe/target hybrid without detecting undue quantities of the unhybridized target probe. This can typically be done using the following procedures:
1. Formation of the target probe/target hybrid on the surface of a solid support followed by segregation of said hybrid away from the unhybridized target probe.

2. Formation of the target probe/target hybrid in solution followed by segregation of said hybrid and said unhybridized target probe.
In order to determine either quantitatively or qualita-tively, the amount of target nucleotide sequence present employing these procedures, the amount of target probe hybridized or unhybridized can be determined and related to the amount of hybrid formed. Typically, the amount of target probe in the hybridized form is determined except in those cases where the target probe displaces a previously hybridized labeled probe (see `for example Clin. Chem. 32, 1696-1701 (1986)).
Thiq method, however, is not limited to hybridization procedures which require a separation step. For example, as improved homogeneous assay methods become available the invention procedure described here will also be useful in homogeneous assay formats .
This invention can be employed in several forms. The requirement being that at some point in the hybridization assay the non-target probe or probes are able to hybridize with the non-target sequence without significantly hybridizing with the target sequence. Such "capping" of the non-target sequence may occur either before, during or after the hybridization between 132'~,5~
the target probe and the target sequence. A preferred embodiment is to employ said "capping" during the hybridization between the target probe and target sequence. This use of the invention can be carried out in several formats. These include:
1. Iso-Thermal. Hybridization occurs at a constant temperature.
2. Slow Cool. The hybridization occurs over a decreasing temperature range generally including the melting temperature (Tm) of the target probe/target nucleotide sequence hybrid.
The non-target probe may, alternatively be added before the target probe to provide for capping of non-target sequences before the target probe is added. It is also suitable to carry out the target probe/target sequence hybridization at high temperatures and/or under high stringency conditions and then add non-target probe to prevent target probe/non-target sequence hybridization as the temperature and/or stringency is lowered.
Preferably, the probes are selected such that the melting temperature of the non-target probe/non-target sequence hybrid, is greater then the melting temperature of the target probe/non-target sequence hybrid. Hybridization conditions are preferably established by first heating the medium to denature at least the probes and any target sequence present (and preferably to a temperature which will denature all nucleotide multimers present), then gradually cooling the medium to a temperature such that the target probe can hybridize with the target sequence (and preferably at which all hybridizations are complete). During cooling, non-target probe can hybridize with non-target sequence to prevent cross reaction when the temperature at which the target probe cross reacts with non-target sequence is 1323~2 subsequently reached. In one aspect of the invention, hybridizing conditions are established by the foregoing means with all polynucleotides present in a liquid medium, absent any solid phase support on which the liquid medium is absorbed.
Such a solid phase would include a solid support such as nitrocellulose.
In another aspect of the invention, the target probe is labelled with one or more atoms or molecules which lend themselves to ready detection. Such labels can include, but are not limited to, radiolabels, enzymes, chemiluminescent labels, colorimetric labels, or fluorescent labels.
Furthermore, the step of testing for the presence of target probe, as already described, preferably involves segregating (and preferably separating) any target probe hybridized with nucleotide multimers, from target probes not so hybridized.
Such segregation (or separation) can be accomplished by selective absorption of polynucleotide hybrids on a solid support such as hydroxyapatite using a procedure such as described in Kohne, D.E. and Britten, R.J., "Nucleic Acid Research" Vol. 2, p. 500-12 (1971). However, where the target nucleotlde sequence is on a polynucleotide of substantially greater length than the target probe (which will be the typical situation where the polynucleotide originates from a clinical sample), separation may be preferably accomplished by selectively immobilizing the longer target sequence and hybridized target probe on polycationic particles ~hrough ionic attraction.
Drawings Embodiments of the invention will now be described with reference to FIGURES 1 and 2.

.

1323~2 FIGURE 1 is a graph illustrating thermal stability of the hybrids formed in the experiment described below.
FIGURE 2 is a diagram illustratinq the relationship of the Tm for various possible hybrids.

Detailed Description of Embodiments of the Invention EXAMPLE I
To demonstrate the present invention, four synthetic oligodeoxynucleotides were prepared, namely "PROBE", "TARGET", "MIS-PROBE", and "MIS-TARGET". The probe is a 35 base sequence which is an exact complement to the "TARGET". MIS-T M GET is the TARGET sequence with two substitutions (mismatches): position 12 has an A substituted for a T and position 24 has a C substituted for a G. MIS-PRO3E i9 the exact complement of MIS-T M GET. The 2 substitutions are placed 1/3 of the distance from the end of the probe. The purine and pyrimidine content is constant among all the sequences as i9 the G + C content.
The probes were are synthesized using an Applied Biosystems, Inc. Model 380A DNA Synthesizer and purified using standard methods ~Tetrahedron, 39 3-22 (1983)).
The sequence of the above oligonucleotides are as follows:

PROBE: S' CAG TCA AAC TCT AGC CAT TAC CTG CTA ~AG TCA TT 3' TARGET: 3' GTC AGT TTG AGA TCG GTA ATG GAC GAT TTC AGT AA S' MIS-PRO9E: S' CAG TCA AAC TCA AGC CAT TAC CTC CTA AAG TCA TT 3' MIS-TARGET: 3' GTC AGT TTG AGT TCG GTA ATG GAG GAT TTC AGT AA S' In the experiments which follow, PROBE and MIS-PROBE act as nucleotide multimer probes, while TARGET and MIS-TARGET each acts as either a target or a non-target nucleotide multimer (the entire sequence of each being either a target or non-target nucleotide sequence).
The thermal stability of each of the probes with each of the target multimers was first investigated. The PROBE and MIS-PROBE were kinase end labeled using T4 kinase 32p and gamma adenosine triphosphate (ATP) and then each was independently hybridized with both the TARGET and MIS-TARGET (for a total of four independent hybridizations) at 50C in 0.48M phosphate buffer pH 6.8, 0.1% sodium dodecyl sulfate (SDS). Aliquots (10 ul) of the solutions were ther. diluted in 3 mls of a "reaction buffer" consisting of 0.1M phosphate buffer pH 6.8 and, 0.1% SDS, and heated for 5 minutes at various temperatures as indicated in FIGURE 1. The percentage of hybridized probe was determined by binding to hydroxyapatite at 45'C in 0.08 M phosphate buffer pH
6.8, 0.02% SDS, using the method of Kohne. supra. The results are plotted in the FIGURE 1 as percent of single stranded nucleotide remaining. The temperature at which half of the probe remains hybridized and half of the probe is single stranded, is known as the melting temperature (Tm) and is us`ed as the measure of thermal stability. When the exact complement (target multimer) of each probe was replaced by the non-target multimer (thus replacing each probes complement by a nucleotide multimer differing in only two bases therefrom) a 6-7C lowering of the Tm resulted.
FIGURE 2 illustrates the hybridization Tm for the various reactions and cross reactions that can occur.
Referring to FIGURE 1, it will be seen that in an assay for either one of TARGET or MIS-TARGET (the one for which the assay is desired being referred to as the "target nucleotide multimer" and the other one being the "non-target nucleotide :

13235~2 multimer"), where both may be present together, it would be difficult to select a temperature at which most of the target multimer would be hybridized with its complimentary probe (the "target probe") without at the same time having a substantial proportion of the target probe hybridized with the non-target multimer. Thus, the non-target nucleotide multimer would interfere with the assay of the target nucleotide multimer, unless a temperature was selected where only a small portion of the targe~ nucleotide multimer is hybridized.
The present invention substantially reduces interference which may occur from a non-target nucleotide sequence, and thus can eliminate the need for stringent temperature control, by having present during hybridization both a target probe and a non-target probe.
To illustrate the foregoing, a further experiment was performed. The PROBE and MIS-PROBE were mixed in reaction buffer, in the molar ratios shown in TABLE 1 below. Using a water bath, the reactions were heated to 70C for 5 minutes and then slowly cooled to 50C over a period of 2 hours. Incubation at 50C was then continued for 1 hour. The initial heating of the reactions assured that all sequences were single stranded (i.e., "denatured") at the start; the final 50C temperature allowed for any combination of probe and target sequence hybridization to occur since it is well below all Tm's. As will be illustrated later, gradual cooling favored the pairing of exact complements over mismatched complements as the temperature slowly decreased through their respective Tm's. Reactions were assayed by binding to hydroxyapatite as described previously.
To obtain the results in TABLE 1(~), the TMGET PROBE
was labeled, and TARGET and MIS-TMGET were the target and non-- 1 ~ 2 3 ~ ~ 2 l79/260 target nucleotide multimers, respectively, while the MIS-PROBE
was unlabeled. The results in Table l(A) show that: (1) hybridization of the target probe and target multimer (reactions A-D) is not unduly reduced by the presence of even up to a 4x excess of non-target probe; (2) excess unlabeled probe under the same conditions competes roughly as might be expected (within the limits of experimental uncertainly) with the labeled probe (reactions E-G); (3) target probe hybridizes significantly with the non-target (reaction H), but this hybridization i9 very effectively removed when even a relatively low amount of non-target probe (1:1 molar ratio with target probe) is present (reactions I-K); and (4) with both probes and both multimers mixed together, the non-target probe apparently hybridizes with non-target multimer so as to reduce the extent of any target probe/non-target multimer hybridization which might occur absent the non-target probe, while hardly affecting the target probe/target multimer hybridization (and certainly not unduly reducing it) (reactions L-0).
Table l~B) shows the results for the same experiment done with mi~-TARGET PROBE as the labeled probe, and T M GET PROBE
as the unlabeled probe. Thus, MIS-TARGET and TARGET can be regarded now as the target and non-target nucleotide multimers, respectively. The results also support conclusions (1) - (4) above. In particular, reactions A and ~ in TABLE lB ~epresent a situation where the two target multimers could not be distin-guished by the single labeled probe (82% vs 78% hybridization).
The presence of the TARGET PROBE (used as a non-target "capping"
probe here) instead promotes a clear difference in hybridization extent. In this case the desired hybridization of the labeled ~3235~2 MIS-PROBE with the MIS-TARGET was 56%, vs 0~ for the labeled MIS-PROBE and TARGET (reactions D and K).
An experiment was done to compare the gradual cool method with the isothermal procedure. Reactions A-D and H-K in Table l(A) were repeated except that the temperature was immedi-ately lowered to an incubation temperature of 50C or 55 C (by placing tubes containing the reaction solutions into water baths of those respective temperatures), as indicated in TABLE 2, after the 70C pre-heating step. Incubation was then continued at the incubation temperature for 2 hours and the reactions were assayed on hydroxyapatite as described previously. The results are shown in TABLE 2. It is apparent from TABLE 2 that when cooling was immediate, the amount of target probe/target multimer hybridiza-tion is somewhat reduced in the presence of the "capping" MIS-TARGET PROBE (reactions A-D), while the amount of T M GET PROBE/
MIS-TARGET multimer hybridization is somewhat increased (reactions H-K). Thus a gradual cooling as part of establishing hybridization, appears to be advantageous in the method of the present invention.

, 13235~2 TABLE 1 ' Effect of Probe Ratios on Competition lA A B _ D E F G H I J K L M _ O
LABELED PROBE l*
UNLABELED PROBE l 2 4 NON-TARGET PROBE l 2 4 l 2 4 l 2 4 .

Target Mis-Target % Hybridization 8470 76 76 31 20 ll 40 5 3 2 8579 71 78 Normalized S Hybridization lOO ô3 90 90 37 2413 48 6 42 lOl 94 85 93 lB 9 8 C D E F G H I J K L _ _ O

UNLABELED MIS PROBE l 2 4 TARGET PR08E l 2 4 l 2 4 l 2 4 Mis-Target Target % Hybridization 8254 54 56 37 22 13 78l l 0 0 8068 56 52 Normalized ~ Hybridization lOO 66 66 68 45 2716 95 13 0 0 98 83 71 o3 * Footnote: Corresponds to O.l p moles of probe in a lO u1 hybridization reaction.

.

1323~2 179/260 Temperature Effect A B C D H I J K
LA8ELED PROBE 1*

"CAPPING" MIS PROBE1 2 4 1 2 4 . _ TarBet Mis-Target % Hybridization 84 70 76 76 405 3 2 ) ) Slow Cool 70 -> 50 Normalized % Hybridization 100 8390 9048 6 4 2 ) % Hybridization 80 56 43 42 45'6 94 ) ) Isothermal 50 Normalized % Hybridization 100 7054 5356 20 11 5 ) % Target Hybridization 80 66 62 5033 9 4 3 ) ) Isothermal 55 Normalized S Hybridization 100 8378 6341 11 5 4 ) * Footnote: Correspond~ to 0.1 p mole of probe in a 10 ~1 hybridization reaction.

1323~2 179/260 It has been illustrated above than, in an assay for a target nucleotide sequence using a target probe, that interference from a non-target nucleotide sequence (typically on another nucleotide multimer) can be reduced by providing a non-target probe which forms a hybrid with the non-target nucleotide multimer with a higher Tm than the Tm for the target probe/non-target probe which forms a hybrid with the non-target nucleotide multimer hybrid. Thus, in an assay for any given target nucleo-tide sequence, potential interference from a closely related non-target nucleotide sequence can be reduced. Furthermore, as has been shown, if hybridizing conditions are established by gradual cooling, the foregoing beneficial effect is further enhanced.
The ratio of non-target probe to target probe can vary substantially. It is preferred that the ratio is no greater than 9 to 1 and the particularly preferred ratio is no greater than 4 to 1.
The amount of non-target probe used can vary over a wide range and yet provide the improved results described above.
~owever, it is preferred that the non-target probe be present in a molar amount about equal equal to the amount of non-target sequence and it is particularly preferable in a molar amount which is in excess of the non-target sequence present in the sample. This can be accomplished by using an amount of probe which greater than the amount of target and non-target RNA
sequences in the sample.
Although in the above experiments the target and non-target nucleotide sequences consisted of the entire length of the target and non-target nucleotide multimers, it will be apparent that this need not necessarily be so. In fact, in moRt clinical applications both sequences will be only a portion of the length _ 132'~552 of substantially longer target and non-target polynucleotides.

Illustration of such a clinical assay is the following example.

EXAMPLE I I
In this case the synthetic target probe and non-target probe were hybridized to cellular RNA, as would be the case in a clinical assay. This system consisted of Neisseria gonorrhoeae (gon RNA as the desired target) and Neisseria meningitidis (men RNA as the undesired mismatched target). Probes were synthesized which were complementary to both RNA species except for a two base mismatch as in EXAMPLE 1. The probe sequences were as follows:
gon probe: 5' GAG GAT TCC GCA CAT GTC AAA ACC AGG TAA 3' men probe: 5' GAG GAT TCC GTA CAT GTC AAG ACC AGG TAA GG 3' The probes were synthesized as in Example I. Probe sequences are obtained by doing sequence alignments of closely related organisms and selecting regions which are specific to the desired organism.
The Tm's of each combination of probe and target RNA
were determined: gon probe plus gon RNA 59.2C, gon probe plus men RNA 48.2C, men probe plus men RNA 61.5C and men probe plus gon RNA 55C. As was the case in the first working example abovè, exact match hybridization had a TM approximately 6-10 above the corresponding mismatched hybridizations.
Hybridization assays were performed which were analogous to the isothermal 55C condition of Table 2 with an additional slow cooling step from 55C-->40C. (In this case hybridizations were done in 0.48M phosphate buffer pH 6.8, 0.5% SDS and the hydroxyapatite separations were done in 0.12M phosphate buffer pH
6.8, 0.02% SDS at 40C.) The reactions were heated at 70C for 5 13235~2 minutes to ensure complete denaturation of all components, immediately cooled to 55C and incubated 15 hours, slow cooled to 40C over a period of 4 hours and then incubation at 40C
continued for another hour. These reactions were incubated longer than in the first example and with a larger excess of probe since these probes were known to hybridize slower.
The results in Table 3 show the same capping effect of the non-target probe (men) on the non-target RNA as was seen in the first example. Hybridization of target probe to target RNA
was not seriously decreased in the presence of a two-fold excess of non-target probe (reaction D), while the undesired cros~
hybridization of target probe to non-target RNA, though small, was eliminated completely at a 1:1 ratio of non-target capping probe to target probe (reaction J).

Table 3 A B C D H I J K
gon target probe 3* 3 3 3 3 3 3 3 men non-target probe 2 3 6 2 3 6 gon target (RNA) men non-target (RNA) of maximum possible hybridization 53 48 38 52 10 1 0 0 Normalized % of maximum possible hybridization 100 91 72 98 19 2 0 0 *Footnote: corresponds to 0.3 p mole of probe in 30 microliter hybridization reaction.
EXAMPLE III

Another experiment was done to determine the effective-ness of capping, using large probe excesses and low concentra-tions of RNA - as might be typical in a clinical assay. In this example the probes and RNA's were mixed in the ratios indicated (in 0.48M phosphate buffer pH 6.8, 0.5% SDS, lmM EDTA, 1 mM

.
, 1 3 2 c~ 5 ~ 2 179/260 EGTA), heated at 70C for 5 minutes, slow cooled at 55C over a period of 1~ hours, incubated at 55C for 2 hours, slow cooled to 45C over a period of 30 minutes and then incubated at 45C for 30 minutes. Hybridization were then separated on hydroxyapatite as above. This incubation scheme allowed for maximum discrimina-tion of exact match hybridizations since there was slow cooling above the intermediate (stringent) temperature of 55C and a prolonged incubation at that temperature.
As seen in Table 4 the capping (non-target) probe again completely eliminated the cross reaction of target probe and non-target RNA while decreasing the specific hybridization (target probe/target RNA) only 26~.

~ ~235~2 179/260 Table 4 gon probe 15* 15 15 15 men probe 30 30 gon RNA
men RNA

% of maximum possible hybridization 129 95 5 o Normalized % of maximum possible hybridization 100 74 4 0 Footnote: corresponds to 0.2 p mole of probe in 100 microliter hybridization reaction.
It will also be of course appreciated from the foregoing that the target probe could be labeled by means of many of the labels well known in the art. Furthermore, a kit can be assembled which contains either separately, or in admixture, the target and non-target probes, for use in ascertaining the presence of a target nucleotide sequence in a medium which may also have present a related non-target nucleotide sequence.
As will be evident to those skilled in the art, various modifications and alterations to the embodiments described above are possible. Accordingly, the scope of the present invention is not limited to the embodiments described in detail above.

Claims

1. An assay for a target nucleotide sequence in a test sample which may contain a closely related non-target nucleotide sequence or sequences, comprising:
(a) contacting any test sample with a target probe and at least one non-target probe under conditions to permit hybridization between the target probe and said target nucleotide sequence while minimizing any undesired hybridization between the target probe and said non-target nucleotide sequence, any said latter undesired hybridization being reduced by the presence of the non-target probe which is capable of hybridizing with any non-target nucleotide sequence present in at least one of the assay steps so as to increase the target probe/target nucleotide sequence to target probe/non-target nucleotide sequence hybridization ratios wherein the hybridization conditions are established with all the nucleotides present in a liquid medium, absent any solid phase support; and, (b) assaying for hybridization of said target probe to any test sample nucleotide sequence.

2. A method as defined in claim 1 wherein the probes are selected such that the melting temperature of the non-target probe/non-target sequence hybrid is greater than the melting temperature of the target probe/non-target sequence hybrid.

3. A method as defined in claim 1 wherein hybridizing conditions are established by first heating the medium to denature at least the probes and any target sequence present, then cooling the medium with an external heat exchange medium the temperature of which is gradually decreased to a temperature at which the target probe can hybridize with the target sequence.

4. A method as defined in claim 1 wherein hybridization occurs at a fixed temperature.

5. A method as defined in any one of claims 1 to 4 wherein the target probe is labelled, the molar ratio of unlabelled probe to labelled probe is no greater than 9 to 1.

6. A method as defined in any one of claims 1 to 4 wherein the molar ratio of non-target probe to target probe is no greater than 4 to 1.

7. An assay in a liquid medium for a target nucleotide sequence in a test sample which may contain a closely related non-target nucleotide sequence comprising:

(a) contacting any test sample in a liquid medium with a target probe and at least one non-target probe under conditions to permit hybridization between the target probe and said target nucleotide sequence while minimizing any undesired hybridization between the target probe and said non-target nucleotide sequence any said latter undesired hybridization being reduced by the presence of the non-target probe which is capable of hybridizing with any non-target nucleotide sequence present in at least one of the assay steps so as to increase the target probe/target nucleotide sequence to target probe/non-target nucleotide sequence hybridization ratios wherein the hybridizing conditions are established with all the nucleotide present in the liquid medium, absent any solid phase support on which the liquid medium is absorbed; and, (b) assaying for hybridization of said target probe to any test sample nucleotide sequence.

8. A method as defined in claim 7 wherein the probes are selected such that the melting temperature of the non-target probe/non-target multimer hybrid is greater than the melting temperature of the target probe/non-target multimer hybrid.

9. A method as defined in claim 7 wherein the probes are selected such that the non-target probe/non-target multimer hybrid has a greater number of complementary nucleotides than the target probe/non-target multimer hybrid.

10. A method as defined in claim 7 wherein hybridizing conditions are established by first heating the liquid medium to denature at least the probe and any target multimer present, then gradually cooling the liquid medium so that the target probe can hybridize with target multimer.

11. A method as defined in claim 7 wherein hybridizing conditions are established by first heating the medium to a temperature which will denature at least the target probe and any target multimer, then cooling the medium with an external heat exchange medium the temperature of which is gradually decreased to a temperature at which the target probe can hybridize with the target sequence.

12. A method as defined in claim 7, 8, 9, 10 or 11 wherein the molar ratio of non-target probe to a target probe is no greater than 9 to 1.

13. A method as defined in claim 7, 8, 9, 10 or 11 wherein the molar ratio of non-target probes to target probes is no greater than 4 to 1.

14. A method as defined in claim 10 wherein the probes are oligonucleotides with some region in common.

15. A method as defined in claim 7 wherein the target multimer is from the group consisting of RNA and DNA.

16. A method as defined in claim 7, 8 or 11 wherein the assaying step comprises first segregating hybrids from unhybridized labelled probe, then identifying the presence of label in the hybrids.

17. A method as defined in claim 7 wherein hybridization occurs at a fixed temperature.

18. A method according to claim 3 or 11 wherein the medium is first heated to about 70°C and is then slowly cooled to 50°C over a period of about two hours.

19. A method according to claim 3 or 11 wherein on cooling the medium, said medium is held at an intermediate temperature for a time sufficient to improve discrimination of exact match hybridizations.

20. A kit useful for assaying a target multimer in a test sample which may also contain a non-target multimer, comprising:

(a) a labelled, nucleotide multimer target probe selected such that under hybridizing conditions, it can form a target probe/target sequence hybrid, but which can also incidentally form a target probe/non-target sequence hybrid;
(b) an unlabelled, nucleotide multimer non-target probe, distinct from the target probe, selected such that under hybridizing conditions, it can form a non-target probe/non-target multimer hybrid so as to reduce the amount of target probe/non-target multimer hybrid which may form absent the non-target probe, while not unduly reducing the amount of target probe/target multimer hybrid which can form wherein the molar ratio of non-target probe to target probe is no greater than 9:1.

21. A kit as defined in claim 20 wherein the target and non-target probes have respective nucleotide sequences with some region in common.

22. A kit as defined in claim 21 wherein the probes are selected such that the melting temperature of the non-target probe/non-target sequence hybrid, is greater than the melting temperature of the target probe/non-target sequence hybrid.

23. A kit as defined in claim 21 wherein the probes are selected such that the unlabelled non-target probe/non-target multimer hybrid will have a greater number of complementary nucleotides than the labelled target-probe/non-target multimer hybrid.

24. A kit as defined in claim 23 wherein the probes are oligonucleotides which differ by one to three nucleotides only.

25. A kit as defined in claim 22, 23 or 24 wherein the molar ratio of non-target probe to target probe is no less than 1 to 1.

26. A kit as defined in claim 22, 23 or 24 wherein the molar ratio of non-target probe to target probe is no greater than 4 to 1.

27. A kit as defined in claim 26 wherein the molar ratio of non-target probe to target probe is no less than 1,1.

28. A kit as defined in claim 22, 23, 24 or 27 wherein the probes can hybridize with a polynucleotide from the group consisting of RNA and DNA, such that the kit is useful for assaying one of those polynucleotides.

29. A kit as defined in claim 22, 23, 24 or 27 wherein the label of the labelled probe is selected from a group consisting of radiolabels, enzymes, chemiluminescent labels, colorimetric labels, and fluorescent labels.