This invention relates to the field of nucleic acid hybridization.  In particular, the present invention relates to methods for ranking the relative specificity with which polynucleotide probes hybridize to a nucleic acid sequence.  The invention also relates to methods of identifying and/or designing nucleic acid sequences which hybridize most specifically to a nucleotide sequence of interest. 
The ability to measure abundances of different nucleic acid molecular species in a sample containing many different nucleic acid sequences is a matter of great interest to many researchers.  Presently, assays involving hybridization of nucleic acid molecules to a complementary probe are the only way to detect the presence of a particular sequence or sequences in a complex sample comprising many different nucleic acid sequences.  For example, the nucleotide sequence similarity of a pair of nucleic acid molecules can be distinguished by allowing the nucleic acid molecules to hybride, and following the kinetic and equilibrium properties of duplex formation (see, e.g., Sambrook, J. et al.,  eds.,  1989, Molecular Cloning: A Laboratory Manual, 2nd Ed.,  Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., at pp.  9.47-9.51 and 11.55-11.61; Ausubel et al.,  eds.,  1989, Current Protocols in Molecular Biology, Vol I, Green Publishing Associates, Inc., John Wiley and Sons, Inc., New York, at pp.  2.10.1-2.10.16; Wetmur, J. G., 1991, Critical Reviews in Biochemistry and Molecular Biology 26:227-259; Persson, B. et al.,  1997, Analytical Biochemistry 246:34-44; Albretsen, C. et al.,  1988, Analytical Biochemistry 170:193-202; Kajimura, Y. et al.,  1990, GATA 7:71-79; Young, S. and Wagner, R. W., 1991, Nucleic Acids Research 19:2463-2470; Guo, Z. et al.,  1997, Nature Biotechnology 15:331-335; Wang, S. et al.,  1995, Biochemistry 34:9774-9784; Niemeyer, C. M. et al.,  1998, Bioconjugate Chemistry 9:168-175). 
Some of the most widely used techniques employ oligonucleotide xe2x80x9cprobes,xe2x80x9d (i.e., DNA molecules having a length up to about 100 bases and more typically fewer than about 50 bases) to selectively hybridize to, and thereby identify, nucleic acid sequences in a sample that contain complementary sequences.  Many assays for detecting nucleic acid sequences in a sample comprise binding a set of nucleic acid probes to a solid support, permitting a labeled nucleic acid species to bind to the immobilized nucleic acid, washing off any unbound material, and detecting the bound, labeled sequence.  For example, in blotting assays, such as dot or Southern Blotting, nucleic acid molecules may be first separated, e.g., according to size by gel electrophoresis, transferred and bound to a membrane filter such as a nitrocellulose or nylon membrane, and allowed to hybridize to a single labeled sequence (see, e.g., Nicoloso, M. et al.,  1989, Biochemical and Biophysical Research Communications 159:1233-1241; Vernier, P. et al.,  1996, Analytical Biochemistry 235:11-19).  Other techniques have been developed to study the hybridization kinetics of polynucleotides immobilized in agarose or polyacrylamide gels (see, e.g., Ikuta S. et al.,  1987, Nucleic Acids Research 15:797-811; Kunitsyn, A. et al.,  1996, Journal of Biomolecular Structure and Dynamics 14:239-244; Day, I. N. M. et al.,  1995, Nucleic Acids Research 23:2404-2412), as well as hybridization to polynucleotide probes immobilized on glass plates (Beattie, W. G. et al.,  1995, Molecular Biotechnology 4:213-225) including oligonucleotide microarrays (Stimpson, D. I. et al.,  1995, Proc.  Natl.  Acad.  Sci.  U.S.A. 92:6379-6383). 
In DNA microarray expression assays, a complex mixture of labeled soluble sequences, derived, e.g., from genes in a population of cells, is analyzed by hybridization to another complex set of sequences which are separated into individual species, each bound separately to a solid support.  The amount of labeled sequence bound to each sequence on the support is used as a measure of the level of expression of the species in the cells (see, e.g., Schena et al.,  1995, Science 270:467-470; Lockhart et al.,  1996, Nature Biotechnology 14:1675-1680; Blanchard et al.,  1996, Nature Biotechnology 14:1649; Ashby et al.,  U.S. Pat.  No.  5,569,588). 
Equilibrium binding during hybridization of nucleic acids with complementary strands is related to (a) the similarity of the hybridizing sequences, (b) the concentration of the nucleic acid sequences, (c) the temperature, and (d) the salt concentration.  Accordingly, it is well known that although hybridization is very selective for matching sequences, related sequences from other genes or gene fragments which are not perfectly complementary will still hybridize at some level.  For oligonucleotide probes targeted at low-abundance species, or at species with closely related (i.e., homologous) molecular family members, such xe2x80x9ccross-hybridizationxe2x80x9d can significantly contaminate and confuse the results of hybridization to the oligonucleotide probes.  For example, cross-hybridization is a particularly significant concern in the detection of single nucleotide polymorphisms (SNP""s) since the sequence to be detected (i.e., the particular SNP) must be distinguished from other sequences that differ by only a single nucleotide. 
To some extent, cross-hybridization can be limited by regulating the temperature and salt conditions (i.e., the xe2x80x9cstringencyxe2x80x9d) of the hybridization or post-hybridization washing conditions.  For example, xe2x80x9chighly stringentxe2x80x9d wash conditions may be employed so as to destabilize all but the most stable duplexes such that hybridization signals are obtained only from the sequences that hybridize most specifically, and are therefore the most homologous, to the probe.  Exemplary highly stringent conditions comprise, e.g., hybridization to filter-bound DNA in 5xc3x97SSC, 1% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65xc2x0 C., and washing in 0.1xc3x97SSC/0.1% SDS at 68xc2x0 C. (Ausubel et al.,  eds.,  1989, Current Protocols in Molecular Biology, Vol.  I, Green Publishing Associates, Inc., and John Wiley and Sons, Inc., New York, N.Y., at p. 2.10.3).  Alternatively, xe2x80x9cmoderate-xe2x80x9d or xe2x80x9clow-stringencyxe2x80x9d wash conditions may be used to identify sequences which are related, not just identical, to the probe, such as members of a multi-gene family, or homologous genes in a different organism.  Such conditions are well known in the art (see, e.g., Sambrook et al.,  supra; Ausubel, F. M. et al.,  supra).  Exemplary moderately stringent wash conditions comprise, e.g., washing in 0.2xc3x97SSC/0.1% SDS at 42xc2x0 C. (Ausubel et al.,  1989, supra).  Exemplary low-stringency washing conditions include, e.g., washing in 5xc3x97SSC or in 0.2xc3x97SSC/0.1% SDS at room temperature (Ausubel et al.,  1989, supra). 
However, the exact wash conditions that are optimal for any given assay will depend on the exact nucleic acid sequence or sequences of interest, and, in general, must be empirically determined.  There is no single hybridization or washing condition which is optimal for all nucleic acid preparations and sequences.  Indeed, even the most optimized conditions can only partially distinguish between competing sequences, especially when the competing sequences are quite similar, or when some of the competing sequences are present in excess amounts or at high concentrations. 
Other existing techniques to minimize cross-hybridization involve the selection and use of particular oligonucleotide probes that are most specific for a particular target nucleic acid molecule of interest.  For example, multiple different oligonucleotide probes which are complementary to different, distinct sequences of a target nucleic acid may be used (see, e.g., Lockhart et al. ( 1996) Nature Biotechnology 14:1675-1680; Graves et al. ( 1999) Trends in Biotechnology 17:127-134).  In other techniques, the oligonucleotide probe is intentionally mismatched, and its hybridization to (or dissociation from) the target nucleic acid molecule is compared to that of the perfect match oligonucleotide probe so that a cross-hybridization component may be subtracted from the total hybridization signal (see, e.g., Graves et al.,  supra). 
However, the use of techniques such as these generally requires some means for selecting those oligonucleotide sequences which hybridize most specifically to a particular target nucleic acid sequence of interest (i.e., with the least cross-hybridization).  Existing numerical models of hybridization can, in principle, predict specificity given the sequence of an oligonucleotide probe as well as the sequences of all the components in the hybridizing sample and their relative abundances.  However, such numerical models are still too crude to provide reliable results.  Further, necessary inputs to such models such as sequence information, relative abundances, and hybridization conditions are frequently only approximately known if at all.  There is an enormous need, therefore, for empirical methods, by which the most specific oligonucleotides may be chosen among the many possible candidates so that cross-hybridization may be limited. 
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. 
The present invention relates to nucleic acid hybridization.  In particular, the invention provides methods for determining the severity of cross-hybridization to a particular oligonucleotide probe.  The methods of the invention can therefore be used to evaluate, in terms of an objective empirically calculated statistic, the specificity of a particular oligonucleotide probe relative to a xe2x80x9cperfect matchxe2x80x9d reference hybridization.  The methods of the invention can also be used to rank a plurality of oligonucleotide probes, by means of an objective, empirically calculated value or xe2x80x9cmetric,xe2x80x9d according to the relative specificity with which each probe hybridizes to a particular polynucleotide sequence in a sample.  Thus, the methods of the present invention can be used to screen a plurality of oligonucleotide probes so that the most specific probe or probes for a particular polynucleotide sequence may be selected among the many possible candidates. 
For any target polynucleotide sequence (e.g., a particular gene, mRNA, or cDNA sequence of interest) there are generally hundreds of thousands (i.e., xcx9c105) of possible oligonucleotide probes, each of different length and/or sequence position, which could be used to detect the polynucleotide by hybridization.  The present invention provides methods to efficiently select, according to an objective standard, the few, most specific oligonucleotides out of the enormous number of possible candidates.  Thus, by using the methods of this invention, the skilled artisan can reduce the number of probes, e.g., on a microarray, for detecting a particular gene, thereby allowing more genes to be reported with a given number of probes.  The methods and compositions of the invention relate, not only to the evaluation of individual polynucleotides, e.g., individual polynucleotide sequences, but also to the evaluation of sets of polynucleotides which have a particular specificity or a particular degree of complementarity to a particular probe.  Such sets of polynucleotides are referred to herein as xe2x80x9cpolynucleotide sets.xe2x80x9d
The invention is based, at least in part, on the discovery that the difference in the integral (i.e., the area) between an actual dissociation curve and a reference dissociation curve is a monotonic function of the level of non-specific hybridization in the actual dissociation curve.  Thus, for example, if a given reference dissociation curve represents hybridization to a particular probe with 100% specificity (i.e., hybridization with zero mismatches), then the area between this reference dissociation curve and an actual dissociation curve obtained for the particular probe indicates the level of non-specific or cross-hybridization to the particular probe (i.e., in the actual dissociation curve). 
The present invention therefore provides methods and compositions which can be used to determine the level or extent of cross-hybridization to a probe.  Specifically, and in more detail, the invention provides, in a first embodiment, a method for determining the specificity with which polynucleotide molecules hybridize to molecules of a given probe.  The methods comprise comparing a dissociation curve representing dissociation of polynucleotide molecules from molecules of the given probe to a reference dissociation curve representing dissociation of the polynucleotide molecules from molecules of a reference probe.  In one particular aspect of this first embodiment, the comparing of the dissociation curve to the reference dissociation curve comprises determining the value of a metric representing the difference between the dissociation curve and the reference dissociation curve, e.g., by subtracting the integral of the dissociation curve from the integral of the reference dissociation curve. 
In a particular aspect of this first embodiment, the dissociation curve is provided by a method comprising: (a) contacting a polynucleotide sample to one or more molecules of the given probe under conditions which allow polynucleotide molecules in the polynucleotide sample to hybridize to the one or more molecules of the given probe; and (b) measuring the polynucleotide molecules hybridized to the one or more molecules of given probe over a time period wherein a detectable fraction of the polynucleotide molecules dissociates from the one or more molecules of the given probe.  Methods of this particular aspect of the first embodiment are also provided wherein the step of measuring the polynucleotide molecules hybridized to the one or more molecules of the given probe comprises: (i) repeatedly washing the polynucleotide sample under conditions such that some fraction of the polynucleotide molecules dissociates from the one or more molecules of the given probe; and (ii) measuring the polynucleotide molecules that remain hybridized to the one or more molecules of the given probe after each washing. 
In another particular aspect of the first embodiment of the invention, the reference dissociation curve is provided by a method comprising: (a) contacting a polynucleotide sample to one or more molecules of the reference probe sequence under conditions which allow polynucleotide molecules in the polynucleotide sample to hybridize to the one or more molecules of the reference probe sequence; and (b) measuring polynucleotide molecules hybridized to the one or more molecules of the reference probe over a time period wherein a detectable fraction of the particular polynucleotide molecules dissociates from the one or more molecules of the reference probe.  Preferably, the reference probe is identical to the given probe in these methods.  However, the invention also provides preferred aspects of the first embodiment wherein the reference probe is chosen to have a binding energy for a perfect match duplex which is similar to or identical to the binding energy of the given probe for a perfect match duplex. 
The invention further provides aspects of the first embodiment wherein the polynucleotide molecules are differentially labeled, e.g., with a fluorescent dye such as fluorescein, rhodamine, texas red; with a fluorescent label such as FAM, JOE, ROX, HEX, TET, IRD40, IRD41, a cyamine dye (e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 or FLUORX), a BODIPY dye (e.g., BODIPY-FL, BODIPY-TR, BODIPY-TMR, BODIPY-630/650, or BODIPY-650/670), or an ALEXA dye (e.g., ALEXA-488, ALEXA-532, ALEXA-546, ALEXA-568 or ALEXA-594); with a radioactive isotope such as 32P, 35S, 14C or 125I; an electron rich molecule, such as ferritin, hemocyanin or colloidal gold; or with a first chemical group specifically complexed to the polynucleotide molecule, and wherein the first chemical group is detected by a method comprising contacting the first chemical group (e.g., avidin or streptavidin) with a second chemical group (e.g., biotin or iminobiotin) that (i) has binding affinity for the first chemical group, and (ii) is covalently linked to an indicator molecule. 
Various aspects of the invention are provided wherein the polynucleotide molecules are naturally occurring polynucleotide molecules such as genomic DNA molecules isolated from cells or from an organism, or RNA molecules isolated from cells or from an organism.  Aspects of the invention are also provided wherein the polynucleotide molecules are, e.g., RNA molecules expressed by a cell or organism (e.g., messenger RNA molecules), cDNA molecules derived therefrom or cRNA molecules derived therefrom.  Aspects of the invention are further provided wherein the polynucleotide molecules are, e.g., synthetic nucleic acid molecules, such as cDNA or a cRNA molecules, or polynucleotide molecules synthesized by polymerase chain reaction.  Aspects of the invention are also provided wherein the polynucleotide molecules comprise short polynucleotide molecules which are representative of a nucleic acid population of a cell. 
In various aspects of the invention the probes are complementary, e.g., to a DNA sequence such as a genomic DNA sequence or a cDNA sequence, or to an RNA sequence such as a messenger RNA sequence or a cRNA sequence.  Various aspects of the invention are also provided wherein the probe comprises a sequence of DNA analogues, a sequence of |RNA analogues, STS""s or SNP""s. 
Various aspects of the invention are further provided wherein the probe or probes are immobilized on a solid support or surface (e.g., a nylon membrane, a cellulose filter or a glass surface).  In fact, a particularly preferred aspect of the invention is provided wherein the probe is part of an array of probes such as a microarray.  In various aspects of the invention provided herein the microarray comprises polynucleotides that are binding sites for fewer than 50% of the genes in the genome of an organism or, alternatively, for at least 50%, at least 75%, at least 85%, at least 90%, or at least 99% of the genes in the genome of an organism.  In various aspects of the invention provided herein the probe of the microarray comprises a polynucleotide sequence of between 200 and 50,000 bases in length or between 300 and 1,000 bases in length, or a single stranded polynucleotide sequence of between 4 and 200 bases in length, between and 150 bases in length, less than 40 bases in length (e.g. between 15 and bases in length), between 40 and 80 bases in length, between 40 and 70 bases in length, and between 50 and 60 bases in length.  In various embodiments provided herein the microarray can comprise at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 50,000, or at least 55,000 different probes per 1 cm2. 
In still other embodiments described herein, the invention provides methods for comparing the specificity with which molecules of a first polynucleotide sequence hybridize to a probe to the specificity with which molecules of a second polynucleotide sequence hybridize to the probe.  The method comprises determining the value of a metric by comparing a first dissociation curve representing dissociation of molecules of the first polynucleotide sequence from the probe to a second dissociation curve representing dissociation of molecules of the second polynucleotide sequence from the probe, wherein the metric is related to the specificity with which molecules of the first polynucleotide sequence hybridizes to the probe relative to molecules of the second polynucleotide sequence. 
In yet other embodiments described herein, the invention provides methods for ranking two or more polynucleotide sequences by the specificity with which molecules of each of the two or more polynucleotide sequences hybridize to a probe.  The method comprises ranking the two or more polynucleotide sequences according to values of a metric, wherein a value of the metric is determined from each of the two or more polynucleotide sequences by a method comprising comparing a dissociation curve representing dissociation of molecules of one of the two or more polynucleotide sequences from molecules of the probe to a reference dissociation curve representing dissociation of molecules of a reference polynucleotide sequence from molecules of the probe, and wherein the value of the metric for each of the two or more polynucleotide sequences is related to the specificity with which molecules of each of the two or more polynucleotide sequences hybridize to molecules of the probe. 
In still other embodiments provided herein, the invention provides a method for comparing the specificity with which molecules of a polynucleotide sequence hybridize to molecules of a first probe to the specificity with which molecules of the polynucleotide sequence hybridize to molecules of a second probe.  The method comprises determining the value of a metric by comparing a first dissociation curve representing dissociation of molecules of the polynucleotide sequence from molecules of the first probe to a second dissociation curve representing dissociation of molecules of the polynucleotide sequence from molecules of the second probe, wherein the metric is related to the specificity with which molecules of the polynucleotide sequences hybridize to the molecules of the first probe relative to the molecules of the second probe. 
In other embodiments, the invention provides methods for ranking two or more probes by the specificity with which molecules of a polynucleotide sequence hybridize to molecules of each of the two or more probes.  The methods comprise ranking the two or more probes according to values of a metric, wherein the value of the metric is determined for each of the two or more probes by a method comprising comparing a dissociation curve representing dissociation of molecules of the polynucleotide sequence from molecules of one of the two or more probes to a reference dissociation curve representing dissociation of molecules of the polynucleotide sequence from molecules of a reference probe, and wherein the value of the metric for each of the two or more probes is related to the specificity with which molecules of the polynucleotide sequence hybridize to molecules of each of the two or more probes. 
In yet other embodiments, the invention provides computer systems that may be used to practice each of the above-described methods of the invention.  Specifically, the invention provides various computer systems comprising a processor and a memory coupled to the processor and encoding one or more programs.  The one or more programs encoded by the memory cause the processor to perform the methods of the invention.  For example, in one embodiment the invention provides a computer system for determining the specificity with which polynucleotide molecules hybridize to molecules of a given probe.  Specifically, in this first embodiment the programs cause the processor to perform a method comprising: (a) comparing a dissociation curve representing dissociation of polynucleotide molecules from molecules of the given probe to a reference dissociation curve representing dissociation of the polynucleotide molecules from a reference probe; and (b) determining the value of a metric from said comparing, wherein the metric represents the difference between the dissociation curve and the reference dissociation curve. 
In another embodiment, the invention provides a computer system for comparing the specificity with which a first polynucleotide sequence hybridizes to a probe to the specificity with which a second polynucleotide sequence hybridizes to said probe.  The computer system comprises a processor and a memory encoding one or more programs coupled to the process.  The one or more programs cause the processor to perform a method comprising: (a) comparing a first dissociation curve representing dissociation of the first polynucleotide sequence from the probe to a second dissociation curve representing dissociation of the second polynucleotide sequence from the probe; and (b) determining the value of a metric from said comparison, wherein the metric represents the difference between the dissociation curve and the reference dissociation curve. 
The invention also provides a computer system for comparing the specificity with which molecules of a first polynucleotide sequence hybridize to molecules of a probe to the specificity with which molecules of a second polynucleotide sequence hybridize to molecules of said probe.  The computer system comprises a processor and a memory encoding one or more programs coupled to the processor.  The one or more programs cause the processor to perform a method comprising: (a) comparing a first dissociation curve representing dissociation of molecules of the first polynucleotide sequence from molecules of the probe to a second dissociation curve representing dissociation of molecules of the second polynucleotide sequence from molecules of the probe; and (b) determining the value of a metric from said comparing, wherein the metric represents the difference between the first dissociation curve and the second dissociation curve. 
The invention also provides a computer system for ranking two or more polynucleotide sequences according to the specificity with which molecules of each of the two or more polynucleotide sequences hybridize to molecules of a probe.  The computer system comprises a processor, and a memory coupled to the processor and encoding one or more programs.  The one or more programs cause the processor to perform a method comprising: (a) comparing each of two or more dissociation curves (each of the two or more dissociation curves representing dissociation of molecules of one of the two or more polynucleotide sequences from molecules of the probe) to a reference dissociation curve representing dissociation of molecules of a reference polynucleotide sequence from molecules of the probe; (b) determining the value of a metric for each of the two or more polynucleotide sequences from each of said comparings, the value of said metric for each of the two or more polynucleotide sequences representing the difference between each of the two or more dissociation curves and the reference dissociation curve; and (c) ranking the two or more polynucleotide sequences according to the value of the metric for each of the two or more polynucleotide sequences. 
The invention also provides a computer system for comparing the specificity with which molecules of a polynucleotide sequence hybridizes to molecules of a first probe relative to the specificity with which molecules of said polynucleotide sequence hybridizes to molecules of a second probe.  The computer system comprises a processor and a memory encoding one or more programs coupled to the processor.  The one or more programs cause the processor to perform a method comprising: (a) comparing a first dissociation curve representing dissociation of molecules of the polynucleotide sequence from molecules of the first probe to a second dissociation curve representing dissociation of molecules of the polynucleotide sequence from molecules of the second probe; and (b) determining the value of a metric from said comparing, wherein the metric represents the difference between the first dissociation curve and the second dissociation curve. 
The invention further provides a computer system for ranking two or more probes by the specificity with which molecules of a polynucleotide sequence hybridize to molecules of each of the two or more probes.  The computer system comprises a processor and a memory encoding one or more programs coupled to the processor.  The one or more programs cause the processor to perform a method comprising: (a) comparing each of two or more dissociation curves (each of the two or more dissociation curves representing dissociation of molecules of the polynucleotide sequence from molecules of one of the two or more probes) to a reference dissociation curve representing dissociation of molecules of the polynucleotide sequence from molecules of the probe; (b) determining the value of a metric for each of the two or more probes from each of said comparings, the value of said metric for each of the two or more probes representing the difference between each of the two or more dissociation curves and the reference dissociation curve; and (c) ranking the two or more probes according to the value of the metric for each of the two or more probes. 
In still other embodiments, the invention provides computer program products for use in conjunction with a computer system (e.g., one of the above-described computer systems of the invention) having a processor and a memory connected to the processor.  The computer program products of the invention comprise a computer readable storage medium having a computer program mechanism encoded or embedded thereon.  The computer program mechanism can be loaded into the memory of the computer and cause the processor to execute the steps of the methods of the invention.  For example, in one aspect of this embodiment, the computer program mechanism can cause the processor to execute the steps of: (a) comparing a dissociation curve representing dissociation of polynucleotide molecules from molecules of a given probe to a reference dissociation curve representing dissociation of the polynucleotide molecules from molecules of a reference probe; and (b) determining the value of a metric from said comparing, said metric representing the difference between the dissociation curve and the reference dissociation curve. 
In another aspect, the computer program mechanism can cause the processor to execute the steps of: (a) comparing a first dissociation curve representing dissociation of molecules of a first polynucleotide sequence from molecules of a probe to a second dissociation curve representing dissociation of molecules of a second polynucleotide sequence from molecules of the probe; and (b) determining the value of a metric from said comparing, said metric representing the difference between the first dissociation curve and the second dissociation curve. 
In yet another aspect, the computer program mechanism can cause the processor to execute the steps of: (a) comparing each of two or more dissociation curves (wherein each of the two or more dissociation curves represents dissociation of molecules of one of two or more polynucleotide sequences from molecules of a probe) to a reference dissociation curve representing dissociation of molecules of a reference polynucleotide sequence from molecules of the probe; (b) determining the value of a metric for each of the two or more polynucleotide sequences from each of said comparings, the value of said metrics for each of the two or more polynucleotide sequences representing the difference between each of the two or more dissociation curves and the reference dissociation curve; and (c) ranking the two or more polynucleotide sequences according to the value of the metric for each of the two or more polynucleotide sequences. 
In still another aspect of this embodiment, the computer program mechanism can cause the processor to execute the steps of: (a) comparing a first dissociation curve representing dissociation of molecules of a polynucleotide sequence from molecules of a first probe to a second dissociation curve representing dissociation of molecules of the polynucleotide sequence from molecules of a second probe; and (b) determining the value of a metric from said comparing, said metric representing the difference between the first dissociation curve and the second dissociation curve. 
In yet another aspect of this embodiment, the computer program mechanism can cause the processor to execute the steps of: (a) comparing each of two or more dissociation curves (wherein each of the two or more dissociation curves represents dissociation of molecules of a polynucleotide sequence from molecules of one of two or more probes) to a reference dissociation curve representing dissociation of molecules of the polynucleotide sequence from molecules of the probe; (b) determining the value of a metric for each of the two or more probes from each of said comparings, the value of said metric for each of the two or more probes representing the difference between each of the two or more dissociation curves and the reference dissociation curve; and (c) ranking the two or more probes according to the value of the metric for each of the two or more probes.