AU3001900A

AU3001900A - Multiplex method for nucleic acid detection

Info

Publication number: AU3001900A
Application number: AU30019/00A
Authority: AU
Inventors: Christine Ann Andrews; Trent Gu; James R Hartnett; Daniel Kephart; Donna Leippe; Martin K. Lewis; Michelle Mandrekar; Richard B. Rhodes; John W Shultz; Roy Welch; Keith V Wood
Original assignee: Promega Corp
Current assignee: Promega Corp
Priority date: 1999-02-18
Filing date: 2000-02-18
Publication date: 2000-09-04
Anticipated expiration: 2020-02-18
Also published as: CA2357011A1; AU774085B2; JP2003535568A; WO2000049181A1; EP1155150A1

Description

WO 00/49181 PCT/USOO/04243 Description MULTIPLEX METHOD FOR NUCLEIC ACID DETECTION 5 Technical Field The invention relates to nucleic acid 10 detection. More specifically, the invention relates to the determination of the presence or absence of multiple targeted, predetermined nucleic acid sequences in nucleic acid target/probe hybrids, and the various applications of their detection. 15 Background of the Invention Methods to detect nucleic acids and to 20 detect specific nucleic acids provide a foundation upon which the large and rapidly growing field of molecular biology is built. There is constant need for alternative methods and products. The reasons for selecting one method over another are varied, and 25 include a desire to avoid radioactive materials, the lack of a license to use a technique, the cost or 30 35 WO 00/49181 PCT/USOO/04243 -2 availability of reagents or equipment, the desire to minimize the time spent or the number of steps, the accuracy or sensitivity for a certain application, the ease of analysis, the need to detect multiple 5 nucleic acids in one sample, or the ability to automate the process. The detection of nucleic acids or specific nucleic acids is often a portion of a process rather than an end in itself. There are many applications 10 of the detection of nucleic acids in the art, and new applications are always being developed. The ability to detect and quantify nucleic acids is useful in detecting microorganisms, viruses and biological molecules, and thus affects many fields, including 15 human and veterinary medicine, food processing and environmental testing. Additionally, the detection and/or quantification of specific biomolecules from biological samples (e.g. tissue, sputum, urine, blood, semen, saliva) has applications in forensic 20 science, such as the identification and exclusion of criminal suspects and paternity testing as well as medical diagnostics. Some general methods to detect nucleic acids are not dependent upon a priori knowledge of 25 the nucleic acid sequence. A nucleic acid detection method that is not sequence specific, but is RNA specific is described in U.S. Patent No. 4,735,897, where RNA is depolymerized using a polynucleotide phosphorylase (PNP) in the presence of phosphate or 30 using a ribonuclease. PNP stops depolymerizing at or near a double-stranded RNA segment. Sometimes double WO 00/49181 PCT/USOO/04243 -stranded RNA can occur as a type of secondary structure RNA, as is common in ribosomal RNA, transfer RNA, viral RNA, and the message portion of mRNA. PNP depolymerization of the polyadenylated 5 tail of mRNA in the presence of inorganic phosphate forms ADP. Alternatively, depolymerization using a ribonuclease forms AMP. The formed AMP is converted to ADP with myokinase, and ADP is converted into ATP by pyruvate kinase or creatine phosphokinase. Either 10 the ATP or the byproduct from the organophosphate co reactant (pyruvate or creatine) is detected as an indirect method of detecting mRNA. In U.S. Patent No. 4,735,897, ATP is detected by a luciferase detection system. In the 15 presence of ATP and oxygen, luciferase catalyzes the oxidation of luciferin, producing light that can then be quantified using a luminometer. Additional products of the reaction are AMP, pyrophosphate and oxyluciferin. 20 Duplex DNA can be detected using intercalating dyes such as ethidium bromide. Such dyes are also used to detect hybrid formation. Hybridization methods to detect nucleic acids are dependent upon knowledge of the nucleic 25 acid sequence. Many known nucleic acid detection techniques depend upon specific nucleic acid hybridization in which an oligonucleotide probe is hybridized or annealed to nucleic acid in the sample or on a blot, and the hybridized probes are detected. 30 A traditional type of process for the detection of hybridized nucleic acid uses labeled WO 00/49181 PCT/USOO/04243 -4 nucleic acid probes to hybridize to a nucleic acid sample. For example, in a Southern blot technique, a nucleic acid sample is separated in an agarose gel based on size and affixed to a membrane, denatured, 5 and exposed to the labeled nucleic acid probe under hybridizing conditions. If the labeled nucleic acid probe forms a hybrid with the nucleic acid on the blot, the label is bound to the membrane. Probes used in Southern blots have been labeled with 10 radioactivity, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase and acridinium esters. Another type of process for the detection of hybridized nucleic acid takes advantage of the 15 polymerase chain reaction (PCR). The PCR process is well known in the art (U.S. Patents No. 4,683,195, No. 4,683,202, and No. 4,800,159). To briefly summarize PCR, nucleic acid primers, complementary to opposite strands of a nucleic acid amplification 20 target sequence, are permitted to anneal to the denatured sample. A DNA polymerase (typically heat stable) extends the DNA duplex from the hybridized primer. The process is repeated to amplify the nucleic acid target. If the nucleic acid primers do 25 not hybridize to the sample, then there is no corresponding amplified PCR product. In this case, the PCR primer acts as a hybridization probe. PCR based methods are of limited use for the detection of nucleic acid of unknown sequence. 30 In a PCR method, the amplified nucleic acid product may be detected in a number of ways, e.g.

WO 00/49181 PCT/USOO/04243 -5 incorporation of a labeled nucleotide into the amplified strand by using labeled primers. Primers used in PCR have been labeled with radioactivity, fluorescent dyes, digoxygenin, horseradish 5 peroxidase, alkaline phosphatase, acridinium esters, biotin and jack bean urease. PCR products made with unlabeled primers may be detected in other ways, such as electrophoretic gel separation followed by dye based visualization. 10 Multiplex PCR assays are well known in the art. For example, U.S. Patent No. 5,582,989 discloses the simultaneous detection of multiple known DNA sequence deletions. The technique disclosed therein uses a first set of probes to 15 hybridize to the targets. Those probes are extended if the targets are present. The extension products are amplified using PCR. Fluorescence techniques are also known for the detection of nucleic acid hybrids, U.S. Patent 20 No. 5,691,146 describes the use of fluorescent hybridization probes that are fluorescence-quenched unless they are hybridized to the target nucleic acid sequence. U.S. Patent No. 5,723,591 describes fluorescent hybridization probes that are 25 fluorescence-quenched until hybridized to the target nucleic acid sequence, or until the probe is digested. Such techniques provide information about hybridization, and are of varying degrees of usefulness for the determination of single base 30 variances in sequences. Some fluorescence techniques involve digestion of a nucleic acid hybrid in a 5'->3' WO 00/49181 PCT/USOO/04243 -6 direction to release a fluorescent signal from proximity to a fluorescence quencher, for example, TaqMan" (Perkin Elmer; U.S. Patent No. 5,691,146 and No. 5,876,930). 5 Enzymes having template-specific polymerase activity for which some 3'-+5' depolymerization activity has been reported include E. coli DNA Polymerase (Deutscher and Kornberg, J. Biol. Chem., 244(11) :3019-28 (1969)), T7 DNA Polymerase (Wong et 10 al., Biochemistry 30:526-37 (1991); Tabor and Richardson, J. Biol. Chem. 265: 8322-28 (1990)), E. coli RNA polymerase (Rozovskaya et al., Biochem. J. 224:645-50 (1994)), AMV and RLV reverse transcriptases (Srivastava and Modak, J. Biol. Chem. 15 255: 2000-4 (1980)), and HIV reverse transcriptase (Zinnen et al., J. Biol. Chem. 269:24195-202 (1994)). A template-dependent polymerase for which 3' to 5' exonuclease activity has been reported on a mismatched end of a DNA hybrid is phage 29 DNA 20 polymerase (de Vega, M. et al. EMBO J., 15:1182-1192, 1996). A variety of methodologies currently exist for the detection of single nucleotide polymorphisms (SNPs) that are present in genomic DNA. SNPs are DNA 25 point mutations or insertions/deletions that are present at measurable frequencies in the population. SNPs are the most common variations in the genome. SNPs occur at defined positions within genomes and can be used for gene mapping, defining population 30 structure, and performing functional studies. SNPs are useful as markers because many known genetic WO 00/49181 PCT/USOO/04243 -7 diseases are caused by point mutations and insertions/deletions. Some SNPs are useful as markers of other disease genes because they are known to cosegregate. 5 In rare cases where an SNP alters a fortuitous restriction enzyme recognition sequence, differential sensitivity of the amplified DNA to cleavage can be used for SNP detection. This technique requires that an appropriate restriction 10 enzyme site be present or introduced in the appropriate sequence context for differential recognition by the restriction endonuclease. After amplification, the products are cleaved by the appropriate restriction endonuclease and products are 15 analyzed by gel electrophoresis and subsequent staining. The throughput of analysis by this technique is limited because samples require processing, gel analysis, and significant interpretation of data before SNPs can be accurately 20 determined. Single strand conformational polymorphism (SSCP) is a second technique that can detect SNPs present in an amplified DNA segment (Hayashi, K. Genetic Analysis: Techniques and Applications 9:73 25 79, 1992). In this method, the double stranded amplified product is denatured and then both strands are allowed to reanneal during electrophoresis in non-denaturing polyacrylamide gels. The separated strands assume a specific folded conformation based 30 on intramolecular base pairing. The electrophoretic properties of each strand are dependent on the folded WO 00/49181 PCT/USOO/04243 -8 conformation. The presence of single nucleotide changes in the sequence can cause a detectable change in the conformation and electrophoretic migration of an amplified sample relative to wild type samples, 5 allowing SNPs to be identified. In addition to the limited throughput possible by gel-based techniques, the design and interpretation of SSCP based experiments can be difficult. Multiplex analysis of several samples in the same SSCP reaction is 10 extremely challenging. The sensitivity required in mutation detection and analysis has led most investigators to use radioactively labeled PCR products for this technique. In the amplification refractory mutation 15 system (ARMS, also known as allele specific PCR or ASPCR), two amplification reactions are used to determine if a SNP is present in a DNA sample (Newton et al. Nucl Acids Res 17:2503, 1989; Wu et al. PNAS 86:2757, 1989). Both amplification reactions contain 20 a common primer for the target of interest. The first reaction contains a second primer specific for the wild type product which will give rise to a PCR product if the wild type gene is present in the sample. The second PCR reaction contains a primer 25 that has a single nucleotide change at or near the 3' end that represents the base change that is present in the mutated form of the DNA. The second primer, in conjunction with the common primer, will only function in PCR if genomic DNA that contains the 30 mutated form of genomic DNA is present. This technique requires duplicate amplification reactions WO 00/49181 PCT/US00/04243 -9 to be performed and analyzed by gel electrophoresis to ascertain if a mutated form of a gene is present. In addition, the data must be manually interpreted. Single base extension (GBA*) is a technique 5 that allows the detection of SNPs by hybridizing a single strand DNA probe to a captured DNA target (Nikiforov, T. et al. Nucl Acids Res 22:4167-4175). Once hybridized, the single strand probe is extended by a single base with labeled dideoxynucleotides. 10 The labeled, extended products are then detected using colorimetric or fluorescent methodologies. A variety of technologies related to real time (or kinetic) PCR have been adapted to perform SNP detection. Many of these systems are platform 15 based, and require specialized equipment, complicated primer design, and expensive supporting materials for SNP detection. In contrast, the process of this invention has been designed as a modular technology that can use a variety of instruments that are suited 20 to the throughput needs of the end-user. In addition, the coupling of luciferase detection sensitivity with standard oligonucleotide chemistry and well-established enzymology provides a flexible and open system architecture. Alternative analytical 25 detection methods, such as mass spectroscopy, HPLC, and fluorescence detection methods can also be used in the process of this invention, providing additional assay flexibility. SNP detection using real-time amplification 30 relies on the ability to detect amplified segments of nucleic acid as they are during the amplification WO 00/49181 PCT/USOO/04243 -10 reaction. Three basic real-time SNP detection methodologies exist: (i) increased fluorescence of double strand DNA specific dye binding, (ii) decreased quenching of fluorescence during 5 amplification, and (iii) increased fluorescence energy transfer during amplification (Wittwer, C. et al. Biotechniques 22:130-138, 1997). All of these techniques are non-gel based and each strategy will be briefly discussed. 10 A variety of dyes are known to exhibit increased fluorescence in response to binding double stranded DNA. This property is utilized in conjunction with the amplification refractory mutation system described above to detect the 15 presence of SNP. Production of wild type or mutation containing PCR products are continuously monitored by the increased fluorescence of dyes such as ethidium bromide or Syber Green as they bind to the accumulating PCR product. Note that dye binding is 20 not selective for the sequence of the PCR product, and high non-specific background can give rise to false signals with this technique. A second detection technology for real-time PCR, known generally as exonuclease primers (TaqMan* 25 probes), utilizes the 5' exonuclease activity of thermostable polymerases such as Taq to cleave dual labeled probes present in the amplification reaction (Wittwer, C. et al. Biotechniques 22:130-138, 1997; Holland, P et al PNAS 88:7276-7280, 1991). While 30 complementary to the PCR product, the probes used in this assay are distinct from the PCR primer and are WO 00/49181 PCT/USOO/04243 -11 dually-labeled with both a molecule capable of fluorescence and a molecule capable of quenching fluorescence. When the probes are intact, intramolecular quenching of the fluorescent signal 5 within the DNA probe leads to little signal. When the fluorescent molecule is liberated by the exonuclease activity of Tag during amplification, the quenching is greatly reduced leading to increased fluorescent signal. 10 An additional form of real-time PCR also capitalizes on the intramolecular quenching of a fluorescent molecule by use of a tethered quenching moiety. The molecular beacon technology utilizes hairpin-shaped molecules with an internally-quenched 15 fluorophore whose fluorescence is restored by binding to a DNA target of interest (Kramer, R. et al. Nat. Biotechnol. 14:303-308, 1996). Increased binding of the molecular beacon probe to the accumulating PCR product can be used to specifically detect SNPs 20 present in genomic DNA. A final, general fluorescent detection strategy used for detection of SNP in real time utilizes synthetic DNA segments known as hybridization probes in conjunction with a process 25 known as fluorescence resonance energy transfer (FRET) (Wittwer, C. et al. Biotechniques 22:130-138, 1997; Bernard, P. et al. Am. J. Pathol. 153:1055 1061, 1998). This technique relies on the independent binding of labeled DNA probes on the 30 target sequence. The close approximation of the two probes on the target sequence increases resonance WO 00/49181 PCT/USOO/04243 -12 energy transfer from one probe to the other, leading to a unique fluorescence signal. Mismatches caused by SNPs that disrupt the binding of either of the probes can be used to detect mutant sequences present 5 in a DNA sample. There is a need for alternative methods for the detection of a plurality of nucleic acid hybrids in a single sample. There is a demand for such methods that are highly sensitive. For example 10 methods to determine viral load of multiple viruses in a single sample that are able to reliably detect as few as 10 copies of a virus present in a body, tissue, fluid, or other biological sample would be in high demand. There is a great demand for methods to 15 determine the presence or absence of nucleic acid sequences that differ slightly from sequences that might otherwise be present. There is a great demand for methods to determine the presence or absence of sequences unique to a particular species in a sample. 20 There is also a great demand for methods that are more highly sensitive than the known methods, quantitative, highly reproducible and automatable. It would be beneficial if another method were available for detecting the presence of a 25 sought-after, predetermined target nucleotide sequence or allelic or polynucleotide variant. It would also be beneficial if such a method were operable using a sample size of the microgram to picogram scale. It would further be beneficial if 30 the various methods listed above were capable of providing multiple analyses in a single assay WO 00/49181 PCT/US0O/04243 -13 (multiplex assays). The disclosure that follows provides one such method. Brief Summary of the Invention 5 A method of this invention is used to determine the presence or absence of a plurality of predetermined (known) nucleic acid target sequences in a nucleic acid sample. Such a method utilizes an enzyme that can depolymerize the 3'-terminus of an 10 oligonucleotide probe hybridized to a nucleic acid target sequence to release one or more identifier nucleotides whose presence or absence can then be determined. One embodiment of the invention 15 contemplates a method for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample. Thus, the presence or absence of at least two predetermined nucleic acid target sequence is sought to be 20 determined. This embodiment comprises the following steps. A treated sample is provided that may contain a plurality of predetermined nucleic acid target sequences hybridized with their respective 25 nucleic acid probes that include an identifier nucleotide in the 3'-terminal region. The treated sample is admixed with a depolymerizing amount of an enzyme whose activity is to release one or more identifier nucleotides from the 3'-terminus of a 30 hybridized nucleic acid probe to form a treated reaction mixture. The treated reaction mixture is WO 00/49181 PCT/USOO/04243 -14 maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom. 5 An analytical output is obtained by analyzing for the presence or absence of released identifier nucleotides, preferably such that the probe from which the nucleotide was released is distinguishable. The analytical output indicates the 10 presence or absence of the nucleotide at the predetermined regions of the nucleic acid targets, and, thereby, the presence or absence of the nucleic acid targets. It is contemplated that an analytical 15 output of the methods of the invention can be obtained in a variety of ways. The analytical output can be ascertained by luminescence. In some preferred embodiments, analysis for released 3' terminal region identifier nucleotides comprises the 20 detection of ATP, either by a luciferase detection system (luminescence) or an NADH detection system (absorbance spectroscopy). In particularly preferred embodiments, ATP molecules are formed from the nucleotide triphosphates released by the 25 depolymerizing step by a phosphate transferring step, for example using an enzyme such as NDPK (Nucleotide Diphosphate Kinase) in the presence of ADP. In some embodiments the ATP is amplified to form a plurality of ATP molecules. In the ATP detection embodiments, 30 typically the enzyme (NDPK) for converting nucleotides and added ADP into ATP is present in the WO 00/49181 PCT/USOO/04243 -15 depolymerization reaction, and thus they are denoted as a "one pot" method. In an alternative embodiment, the analytical output is obtained by fluorescence 5 spectroscopy. Use of a wide variety of fluorescence detection methods is contemplated. In one exemplary contemplated method, an identifier nucleotide includes a fluorescent label. In a multiplex analysis where it is desirable to distinguish which 10 nucleic acid target sequences are present and which are absent, multiple types of labels can be used. An identifier nucleotide can be fluorescently labeled prior to, or after, release of the identifier nucleotide. It is also contemplated that other than 15 a released identifier nucleotide contains a fluorescent tag. In such an embodiment, the release of nucleotides in a process of the invention is ascertained by a determination of a difference in the length of the polynucleotide probe, for example by 20 capillary electrophoresis imaged by a fluorescent tag at the 5' terminus of the probe or in a region other than the 3' terminal region. In an alternative embodiment, the analytical output is obtained by mass spectrometry. 25 It is preferred here that an identifier nucleotide be a nucleotide analog or a labeled nucleotide and have a molecular mass that is different from the mass of a usual form of that nucleotide, although a difference in mass is not required. It is also noted that with 30 a fluorescently labeled identifier nucleotide, the analytical output can also be obtained by mass WO 00/49181 PCT/USOO/04243 -16 spectrometry. It is also contemplated that the analysis of released nucleotides be conducted by ascertaining the difference in mass of the probe after a depolymerization step of a process of the 5 invention. In another alternative embodiment, the analytical output is obtained by absorbance spectroscopy. Such analysis monitors the absorbance of light in the ultraviolet and visible regions of 10 the spectrum to determine the presence of absorbing species. In one aspect of such a process, released nucleotides are separated from hybridized nucleic acid and other polynucleotides by chromatography (e.g. HPLC or GC) or electrophoresis (e.g. PAGE or 15 capillary electrophoresis). Either the released identifier nucleotides or the remainder of the probe can be analyzed for to ascertain the release of the identifier nucleotide in a process of the invention. In another aspect of such a process a label may be 20 incorporated in the analyzed nucleic acid. In another contemplated embodiment, a sample to be assayed is admixed with two or more nucleic acid probes under hybridizing conditions to form a hybridization composition. The 3'-terminal 25 region of a nucleic acid probe hybridizes with partial or total complementarity to the nucleic acid target sequence when that sequence is present in the sample. The 3'-terminal region of the nucleic acid probe includes an identifier nucleotide. 30 The hybridization composition is maintained under hybridizing conditions for a time period WO 00/49181 PCT/USOO/04243 -17 sufficient to form a treated sample that may contain said predetermined nucleic acid target sequence hybridized with a nucleic acid probe. The treated sample is admixed with a depolymerizing amount of an 5 enzyme whose activity is to release one or more nucleotides from the 3'-terminus of a hybridized nucleic acid probe to form a treated reaction mixture. The treated reaction mixture is maintained under depolymerizing conditions for a time period 10 sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom. The presence of released identifier nucleotides is analyzed to obtain an analytical 15 output, the analytical output indicating the presence or absence of the nucleic acid target sequence. The analytical output may be obtained by various techniques as discussed above. One method of the invention contemplates 20 interrogating the presence or absence of a specific bases in their nucleic acid target sequences in a sample to be assayed, and comprises the following steps. Here, a hybridization composition is formed by admixing a sample to be assayed with a plurality 25 of nucleic acid probes under hybridizing conditions. The sample to be assayed may contain a nucleic acid target sequence to be interrogated. The nucleic acid target comprises at least one base whose presence or absence is to be identified. The hybridization 30 composition includes a plurality of nucleic acid probes that are each substantially complementary to a WO 00/49181 PCT/USOO/04243 -18 nucleic acid target sequence of interest and each probe comprises at least one predetermined nucleotide at an interrogation position, and an identifier nucleotide in the 3'-terminal region. 5 A treated sample is formed by maintaining the hybridization composition under hybridizing conditions for a time period sufficient for base pairing to occur for all probes when a probe nucleotide at an interrogation position is aligned 10 with a base to be identified in its target sequence. A treated reaction mixture is formed by admixing the treated sample with an enzyme whose activity is to release one or more identifier nucleotides from the 3'-terminus of a hybridized nucleic acid probe to 15 depolymerize the hybrid. The enzymes that can be used in this reaction are further discussed herein. The treated reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize the 20 hybridized nucleic acid and release an identifier nucleotide. An analytical output is obtained by analyzing for the presence or absence of released identifier nucleotides. The analytical output 25 indicates the presence or absence of the specific base or bases to be identified. The analytical output is obtained by various techniques, as discussed herein. Preferably, an identifier nucleotide is at the interrogation position. In one 30 preferred embodiment, one is able to determine if at least one of the plurality of targets is present in WO 00/49181 PCT/USOO/04243 -19 the sample. In an alternative preferred embodiment, one is able to determine which of the plurality of targets are present and which are absent. A method that identifies the particular 5 base present at an interrogation position, optionally comprises a first probe, a second probe, a third probe, and a fourth probe. An interrogation position of the first probe comprises a nucleic acid residue that is a deoxyadenosine or adenosine residue. An 10 interrogation position of the second probe comprises a nucleic acid residue that is a deoxythymidine or uridine residue. An interrogation position of the third probe comprises a nucleic acid residue that is a deoxyguanosine or guanosine residue. An 15 interrogation position of the fourth nucleic acid probe comprises a nucleic acid residue that is a deoxycytosine or cytosine residue. Preferably, all four probes can be used in a single depolymerization reaction, and their released identifier nucleotides 20 are distinguishable. In another aspect of the invention, the sample containing a plurality of target nucleic acid sequences is admixed with a plurality of the nucleic acid probes. Several analytical outputs can be 25 obtained from such multiplexed assays. In a first embodiment, the analytical output obtained when at least one nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is greater than the analytical output when all of the 30 nucleic acid probes hybridize with total complementarity to their respective nucleic acid WO 00/49181 PCT/USOO/04243 -20 target sequences., In a second embodiment, the analytical output obtained when at least one nucleic acid probe hybridizes with partial complementarity to one target nucleic acid sequence is less than the 5 analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences. In a third embodiment, the analytical output obtained when at least one nucleic acid probe hybridizes with total 10 complementarity to one nucleic acid target sequence is greater than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences. In a fourth embodiment, the 15 analytical output obtained when at least one nucleic acid probe hybridizes with total complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their 20 respective nucleic acid target sequences. The depolymerizing enzymes for use in these four embodiments are as described herein. Yet another embodiment of the invention contemplates a method for determining the presence or 25 absence of a first nucleic acid target in a nucleic acid sample that may contain that target or may contain a substantially identical second target. For example, the second target may have a single base substitution, deletion or addition relative to the 30 first nucleic acid target. This embodiment comprises the following steps.

WO 00/49181 PCT/USOO/04243 -21 A sample to be assayed is admixed with a plurality of nucleic acid probes under hybridizing conditions to form a hybridization composition. The first and second nucleic acid targets each comprise a 5 region of sequence identity except for at least a single nucleotide at a predetermined position that differs between the targets. Each of the nucleic acid probes is substantially complementary to a nucleic acid target region of sequence identity and 10 comprises at least one identifier nucleotide at an interrogation position. An interrogation position of the probe is aligned with the predetermined position of a target when a target and probe are hybridized. Each probe also includes an identifier nucleotide in 15 the 3'-terminal region. The hybridization composition is maintained under hybridizing conditions for a time period sufficient to form a treated sample wherein the nucleotide at the interrogation position of the probe 20 is aligned with the nucleotide at the predetermined position in the region of identity of the target. A treated reaction mixture is formed by admixing the treated sample with a depolymerizing amount of an enzyme whose activity is to release one 25 or more nucleotides from the 3'-terminus of a hybridized nucleic acid probe. The reaction mixture is maintained under depolymerization conditions for a time period sufficient to permit the enzyme to depolymerize the hybridized nucleic acid and release 30 the identifier nucleotides.

WO 00/49181 PCT/US0O/04243 -22 An analytical output is obtained by analyzing for the presence or absence of identifier nucleotides released from the 3' terminus of the hybridized probe. The analytical output indicates 5 the presence or absence of released identifier nucleotide at the predetermined region, and; thereby, the presence or absence of a corresponding nucleic acid target. One aspect of the above method is comprised 10 of a first probe and a second probe in the same hybridization composition. The first probe comprises a nucleotide an interrogation position that is complementary to a first nucleic acid target at a predetermined position. The second probe comprises a 15 nucleotide at an interrogation position that is complementary to a second nucleic acid target at a predetermined position. Another aspect of the above method, the presence or absence of a third nucleic acid target, 20 which is different from the first and second targets, is assayed for in the same sample that may further contain a fourth target that is substantially identical to the third target. In one aspect of a process of the 25 invention, the depolymerizing enzyme, whose activity is to release nucleotides, is a template-dependent polymerase, whose activity is to depolymerize hybridized nucleic acid, whose 3'-terminal nucleotide is matched, in the 3' to 5' direction in the presence 30 of pyrophosphate ions to release one or more nucleotides. Thus, the enzyme's activity is to WO 00/49181 PCT/USOO/04243 -23 depolymerize hybridized nucleic acid to release identifier nucleotides under depolymerizing conditions. Preferably, this enzyme depolymerizes hybridized nucleic acids whose bases in the 3' 5 terminal region of the probe are matched with total complementarity to the corresponding bases of the nucleic acid target.. The enzyme will continue to release properly paired bases from the 3'-terminal region and will stop at or near the location where 10 the enzyme arrives at a base that is mismatched. In an alternative aspect of the process, the depolymerizing enzyme, whose activity is to release nucleotides, exhibits a 3' to 5' exonuclease activity in which hybridized nucleic acids having one 15 or more mismatched bases at the 3'-terminus of the hybridized probe are depolymerized. Thus, the enzyme's activity is to depolymerize hybridized nucleic acid to release nucleotides under depolymerizing conditions. In this embodiment, the 20 hybrid may be separated from the free probe prior to enzyme treatment. In some embodiments, an excess of target may be used so that the concentration of free probe in the enzyme reaction is extremely low. In still another alternative aspect of a 25 process of the invention, the depolymerizing enzyme exhibits a 3' to 5' exonuclease activity on a double stranded DNA substrate having one or more matched bases at the 3' terminus of the hybrid. The enzyme's activity is to depolymerize hybridized nucleic acid 30 to release nucleotides containing a 5' phosphate under depolymerizing conditions.

WO 00/49181 PCT/USOO/04243 -24 In a further aspect of the invention, the nucleic acid sample to be assayed is obtained from a biological sample that is a solid or liquid. Exemplary solid biological samples include animal 5 tissues such as those obtained by biopsy or post mortem, and plant tissues such as leaves, roots, stems, fruit and seeds. Exemplary liquid samples include body fluids such as sputum, urine, blood, semen and saliva of an animal, or a fluid such as sap 10 or other liquid obtained when plant tissues are cut or plant cells are lysed or crushed. In one aspect of the method, the predetermined nucleic acid target sequence is a microbial or viral nucleic acid and nucleic acid 15 probes comprise sequences complementary to those microbial or viral nucleic acid sequences. In another aspect of the invention, the predetermined nucleic acid target sequence is a gene or region of a gene that is useful for genomic 20 typing. Exemplary target sequences include the Leiden V mutation, a mutant P-globin gene, the cystic fibrosis-related gene in the region of the delta 508 allele, a mutation in a prothrombin gene, congenital adrenal hyperplasia-associated genes, a translocation 25 that takes place in the region of the bcr gene along with involvement of a segment of the abl gene, as well as the loss of heterozygosity of the locus of certain alleles as is found in certain cancers and also allelic trisomy. 30 Genomic typing can also be used to assay plant genomes such as that of rice, soy or maize, and WO 00/49181 PCT/US00/04243 -25 the genomes of microbes such as Campylobacter jejuni, Listeria, E. coli 0H157, and the genomes of viruses such as cytomegalovirus (CMV) or human immunodeficiency virus (HIV). 5 A still further embodiment of the invention contemplates a method using thermostable DNA polymerase as a depolymerizing enzyme for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a 10 nucleic acid sample, and comprises the following steps. A treated sample is provided that may contain a plurality of predetermined nucleic acid target sequences hybridized to their respective 15 nucleic acid probes whose 3'-terminal regions are complementary to their predetermined nucleic acid target sequences and include an identifier nucleotide in the 3'-terminal region. A treated depolymerization reaction mixture is formed by 20 admixing a treated sample with a depolymerizing amount of a enzyme whose activity is to release an identifier nucleotide from the 3'-terminus of a hybridized nucleic acid probe. In a preferred one pot embodiment, the depolymerizing enzyme is 25 thermostable and more preferably, the treated reaction mixture also contains (i) adenosine 5' diphosphate, (ii) pyrophosphate, and (iii) a thermostable nucleoside diphosphate kinase (NDPK). The treated sample is maintained under 30 depolymerizing conditions at a temperature of about 4 0 C to about 90 0 C, more preferably at a temperature of WO 00/49181 PCT/USOO/04243 -26 about 20 0 C to about 90 0 C, and most preferably at a temperature of about 25 0 C to about 80'C, for a time period sufficient to permit the depolymerizing enzyme to depolymerize the hybridized nucleic acid probe and 5 release an identifier nucleotide as a nucleoside triphosphate. In preferred one-pot reactions, the time period is also sufficient to permit NDPK enzyme to transfer a phosphate from the released nucleoside triphosphate to added ADP, thereby forming ATP. The 10 presence or absence of a nucleic acid target sequence is determined from the analytical output obtained using ATP. In a preferred method of the invention, analytical output is obtained by luminescence spectrometry. 15 In another aspect of the thermostable enzyme one-pot method for determining the presence or absence of a predetermined nucleic acid target sequence in a nucleic acid sample, the treated sample is formed by the following further steps. A 20 hybridization composition is formed by admixing the sample to be assayed with a plurality of nucleic acid probes under hybridizing conditions. The 3'-terminal region of the nucleic acid probe (i) hybridizes with partial or total complementarity to a nucleic acid 25 target sequence when that sequence is present in the sample, and (ii) includes an identifier nucleotide. A treated sample is formed by maintaining the hybridization composition under hybridizing conditions for a time period sufficient for the 30 predetermined nucleic acid target sequence to hybridize with the nucleic acid probe.

WO 00/49181 PCT/USOO/04243 -27 Preferably, for the thermostable enzyme method, the depolymerizing enzyme is from a group of thermophilic DNA polymerases comprising Tne triple mutant DNA polymerase, Tne DNA polymerase, Taq DNA 5 polymerase, Ath DNA polymerase, Tvu DNA polymerase, Bst DNA polymerase, and Tth DNA polymerase. In another aspect of the method, the NDPK is that encoded for by the thermophilic bacteria Pyrococcus furiosis (Pfu) 10 A still further embodiment of the invention contemplates determining the presence or absence of a plurality of nucleic acid target sequences in a nucleic acid sample using a plurality of special nucleic acid probes. These special probes hybridize 15 to the target nucleic acid and are then modified to be able to form a hairpin structure. This embodiment comprises the following steps. A treated sample is provided that contains a nucleic acid sample that may include a plurality of 20 nucleic acid target sequences, each having an interrogation position, and each hybridized with its respective nucleic acid probe. The probes are comprised of at least two sections. The first section contains the probe 3'-terminal about 10 to 25 about 30 nucleotides. These nucleotides are complementary to the target strand sequence at positions beginning about 1 to about 30 nucleotides downstream of the interrogation position. The second section of the probe is located at the 5'-terminal 30 region of the probe and contains about 10 to about 20 nucleotides of the target sequence. This sequence WO 00/49181 PCT/USOO/04243 -28 spans the region in the target from the nucleotide at or just upstream (5') of the interrogation position, to the nucleotide just upstream to where the 3' terminal nucleotide of the probe anneals to the 5 target. An optional third section of the probe, from zero to about 50, and preferably about zero to about 20 nucleotides in length and comprising a sequence that does not hybridize with either the first or second section, is located between the first and 10 second sections of the probe. The hybridized probes of the treated sample are extended in a template-dependent manner, as by admixture with dNTPs and a template-dependent polymerase, at least through the interrogation 15 position, thereby forming an extended probe/target hybrid. In a preferred embodiment, the length of the probe extension is limited by omission from the extension reaction of a dNTP complementary to a nucleotide of the target sequence that is present 20 upstream of the interrogation position and absent between the nucleotide complementary to the 3'-end of the interrogation position. The extended probe/target hybrids are separated from any unreacted dNTPs. The extended 25 probe/target hybrid is denatured to separate the strands. The extended probe strands are permitted to form hairpin structures. A treated reaction mixture is formed by admixing the hairpin structure-containing composition 30 with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from WO 00/49181 PCT/USOO/04243 -29 the 3'-terminus of an extended probe hairpin structure. The reaction mixture is maintained under depolymerizing conditions for a time period sufficient for the depolymerizing enzyme to release 5 3'-terminus nucleotides, and then analyzed for the presence of released identifier nucleotides. The analytical output indicates the presence or absence of the nucleic acid target sequences. In a preferred embodiment, the analytical output for the various 10 targets are distinguishable. A still further embodiment of the invention, termed REAPER m , also utilizes hairpin structures. This method contemplates determining the presence or absence of a plurality of nucleic acid 15 target sequences, or a specific base within a target sequence, in a nucleic acid sample, and comprises the following steps. A treated sample is provided that contains a nucleic acid sample that may include a plurality of nucleic acid target sequences hybridized 20 with their respective first nucleic acid probe strands. The hybrid is termed the first hybrid. The first probes are comprised of at least two sections. The first section contains the probe 3'-terminal 25 about 10 to about 30 nucleotides that are complementary to the target nucleic acid sequence at a position beginning about 5 to about 30 nucleotides downstream of the target interrogation position. The second section of the first probe contains about 5 to 30 about 30 nucleotides that are a repeat of the target sequence from the interrogation position to about 10 WO 00/49181 PCT/USOO/04243 -30 to about 30 nucleotides downstream of the interrogation position, and does not hybridize to the first section of the probe. An optional third section of the probe, located between the first and 5 second sections of the probe, is zero to about 50, preferably up to about 20, nucleotides in length and comprises a sequence that does not hybridize to either the first or second section. The first hybrid in the treated sample is 10 extended at the 3 -end of the first probes, thereby extending the first probes past the interrogation position and forming an extended first hybrid whose sequence includes an interrogation position. The extended first hybrid is comprised of the original 15 target nucleic acids and extended first probes. The extended first hybrid is then denatured in an aqueous composition to separate the two nucleic acid strands of the hybridized duplexes and form an aqueous solution containing separated target nucleic acids 20 and a separated extended first probes. Second probes that are about 10 to about 2000, preferably about 10 to about 200, most preferably about 10 to about 30 nucleotides in length and is complementary to the extended first probes at 25 a position beginning about 5 to about 2000, preferably about 5 to about 200, nucleotides downstream of the interrogation position in extended first probe, is annealed to the extended first probe, thereby forming the second hybrid. The second 30 hybrids is extended at the 3'-end of the second probes until that extension reaches the 5'-end of the WO 00/49181 PCT/USOO/04243 -31 extended first probes, thereby forming a second extended hybrid whose 3'-region includes an identifier nucleotide. In preferred embodiments the extending polymerase for both extensions does not add 5 a nucleotide to the 3' end that does not have a corresponding complementary nucleotide in the template. An aqueous composition of the extended second hybrid is denatured to separate the two 10 nucleic acid strands. The aqueous composition so formed is cooled to form a "hairpin structure" from the separated extended second probes when its respective target sequence is present in the original nucleic acid sample. 15 A treated reaction mixture is formed by admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3'-terminus of a nucleic acid hybrid. The 20 reaction mixture is maintained under depolymerizing conditions for a time period sufficient to release 3'-terminal region identifier nucleotides, and then analyzed for the presence of released identifier nucleotide. The analytical output indicates the 25 presence or absence of the various nucleic acid target sequences. The present invention has many benefits and advantages, several of which are listed below. One benefit of the invention is that, in 30 some embodiments, a plurality of nucleic acid hybrids can be detected with very high levels of sensitivity WO 00/49181 PCT/USOO/04243 -32 without the need for radiochemicals or electrophoresis. An advantage of the invention is that the presence or absence of a plurality of target nucleic 5 acid(s) can be detected reliably, reproducibly, and with great sensitivity. A further benefit of the invention is that quantitative information can be obtained about the amount of a target nucleic acid sequence present in a 10 sample. A further advantage of the invention is that very slight differences in nucleic acid sequence are detectable, including single nucleotide polymorphisms (SNPs). 15 Yet another benefit of the invention is that the presence or absence of a number of target nucleic acid sequences can be determined in the same assay. Yet another advantage of the invention is 20 that the presence or absence of target nucleic acids can be determined with a small number of reagents and manipulations. Another benefit of the invention is that the processes lend themselves to automation. 25 Still another benefit of the invention is its flexibility of use in many different types of applications and assays including, but not limited to, detection of mutations, translocations, and SNPs in nucleic acid (including those associated with 30 genetic disease), determination of viral load, WO 00/49181 PCT/USOO/04243 -33 species identification, sample contamination, and analysis of forensic samples. Still further benefits and advantages of the invention will become apparent from the 5 specification and claims that follow. Brief Description of the Drawing In the drawings forming a portion of this disclosure, 10 Fig. 1 illustrates the annealing of the 10865 oligonucleotide (SEQ ID NO:82) to 10870 wild type (SEQ ID NO:83) and 10994 mutant (SEQ ID NO:84) oligonucleotides utilized in rolling circle amplification as Fig. 1A and Fig. 1B, respectively. 15 Also shown are the annealing (hybridization) of oligonucleotide 10866 to oligonucleotide 10865, as well as the hybridization of oligonucleotide probe 10869 (SEQ ID NO:85) to oligonucleotide 10870 and of oligonucleotide probe 10989 (SEQ ID NO:86) to 20 oligonucleotide 10994 as representations of the binding of those probes to the respective amplified sequences. Arcuate lines in oligonucleotide 10865 are used to help illustrate the shape that oligonucleotide 10865 can assume when hybridized with 25 either of oligonucleotides 10870 or 10994. Fig. 2. illustrates the Reaper'" assay as illustrated in Example 89. Fig. 2A illustrates the first hybrid formed by the annealing of nucleic acid target SEQ ID NO: 61 (61) to first probe SEQ ID NO: 30 62 (62). An arrow points to an interrogation position in 286.

WO 00/49181 PCT/USOO/04243 -34 Fig. 2B illustrates the first extended hybrid formed by the annealing of 61 to the extended 62. Extended 287 is first extended probe SEQ ID NO: 63 (63). 5 Fig. 2C illustrates the second hybrid formed by annealing of 63 from the denatured nucleic acid molecule shown in Fig. 2B to the second probe denoted SEQ ID NO: 64 (64). An arrow points to the interrogation position in 63. 10 Fig. 2D illustrates the extended second hybrid formed by the annealing of 63 and the extended 64 strand denoted SEQ ID NO: 65 (65). Fig. 2E illustrates the 65 strand denatured from Fig. 2D and forming a hairpin structure. An 15 arrow points to the interrogation position at the 3' terminus of the hybrid. Definitions To facilitate understanding of the 20 invention, a number of terms are defined below. "Nucleoside", as used herein, refers to a compound consisting of a purine [guanine (G) or adenine (A)] or pyrimidine [thymine (T), uridine (U) or cytidine (C)] base covalently linked to a pentose, 25 whereas "nucleotide" refers to a nucleoside phosphorylated at one of its pentose hydroxyl groups. "XTP", "XDP" and "XMP" are generic designations for ribonucleotides and deoxyribonucleotides, wherein the "TP" stands for triphosphate, "DP" stands for 30 diphosphate, and "MP" stands for monophosphate, in conformity with standard usage in the art.

WO 00/49181 PCT/USOO/04243 -35 Subgeneric designations for ribonucleotides are "NMP", "NDP" or "NTP", and subgeneric designations for deoxyribonucleotides are "dNMP", "dNDP" or "dNTP". Also included as "nucleoside", as used 5 herein, are materials that are commonly used as substitutes for the nucleosides above such as modified forms of these bases (e.g. methyl guanine) or synthetic materials well known in such uses in the art, such as inosine. 10 A "nucleic acid," as used herein, is a covalently linked sequence of nucleotides in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the pentose of the next, and in which the 15 nucleotide residues (bases) are linked in specific sequence; i.e., a linear order of nucleotides. A "polynucleotide," as used herein, is a nucleic acid containing a sequence that is greater than about 100 nucleotides in length. An "oligonucleotide," as 20 used herein, is a short polynucleotide or a portion of a polynucleotide. An oligonucleotide typically contains a sequence of about two to about one hundred bases. The word "oligo" is sometimes used in place of the word "oligonucleotide". 25 A base "position" as used herein refers to the location of a given base or nucleotide residue within a nucleic acid. A "nucleic acid of interest," as used herein, is any particular nucleic acid one desires to 30 study in a sample.

WO 00/49181 PCT/USOO/04243 -36 The term "isolated" when used in relation to a nucleic acid or protein, refers to a nucleic acid sequence or protein that is identified and separated from at least one contaminant (nucleic acid 5 or protein, respectively) with which it is ordinarily associated in its natural source. Isolated nucleic acid or protein is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids or 10 proteins are found in the state they exist in nature. As used herein, the term "purified" or "to purify" means the result of any process which removes some contaminants from the component of interest, such as a protein or nucleic acid. The percent of a 15 purified component is thereby increased in the sample. The term "wild-type," as used herein, refers to a gene or gene product that has the characteristics of that gene or gene product that is 20 most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant" as used herein, refers to a gene or gene product that displays modifications in sequence 25 and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Nucleic acids are known to contain different types of mutations. As used herein, a 30 "point" mutation refers to an alteration in the sequence of a nucleotide at a single base position.

WO 00/49181 PCT/USOO/04243 -37 A "lesion", as used herein, refers to site within a nucleic acid where one or more bases are mutated by deletion or insertion, so that the nucleic acid sequence differs from the wild-type sequence. 5 A "single nucleotide polymorphism" or SNP, as used herein, is a variation from the most frequently occurring base at a particular nucleic acid position. Homologous genes or alleles from different 10 species are also known to vary in sequence. Regions of homologous genes or alleles from different species can be essentially identical in sequence. Such regions are referred to herein as "regions of identity." It is contemplated herein that a "region 15 of substantial identity" can contain some "mismatches," where bases at the same position in the region of identity are different. This base position is referred to herein as "mismatch position." DNA molecules are said to have a "5' 20 terminus" (5' end) and a "3'-terminus" (3' end) because nucleic acid phosphodiester linkages occur to the 5' carbon and 3' carbon of the pentose ring of the substituent mononucleotides. The end of a polynucleotide at which a new linkage would be to a 25 5' carbon is its 5' terminal nucleotide. The end of a polynucleotide at which a new linkage would be to a 3' carbon is its 3' terminal nucleotide. A terminal nucleotide, as used herein, is the nucleotide at the end position of the 3'- or 5'-terminus. As used 30 herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, also can WO 00/49181 PCT/USOO/04243 -38 be said to have 5'- and 3'- ends. For example, a gene sequence located within a larger chromosome sequence can still be said to have a 5'- and 3'-end. As used herein, the 3'-terminal region of 5 the nucleic acid probe refers to the region of the probe including nucleotides within about 10 residues from the 3'-terminal position. In either a linear or circular DNA molecule, discrete elements are referred to as being 10 "upstream" or "5'" relative to an element if they are bonded or would be bonded to the 5'-end of that element. Similarly, discrete elements are "downstream" or "3'" relative to an element if they are or would be bonded to the 3'-end of that element. 15 Transcription proceeds in a 5' to 3' manner along the DNA strand. This means that RNA is made by the sequential addition of ribonucleotide-5' triphosphates to the 3'-terminus of the growing chain (with the elimination of pyrophosphate). 20 As used herein, the term "target nucleic acid" or "nucleic acid target" refers to a particular nucleic acid sequence of interest. Thus, the "target" can exist in the presence of other nucleic acid molecules or within a larger nucleic acid 25 molecule. As used herein, the term "nucleic acid probe" refers to an oligonucleotide or polynucleotide that is capable of hybridizing to another nucleic acid of interest. A nucleic acid probe may occur 30 naturally as in a purified restriction digest or be produced synthetically, recombinantly or by PCR WO 00/49181 PCT/USOO/04243 -39 amplification. As used herein, the term "nucleic acid probe" refers to the oligonucleotide or polynucleotide used in a method of the present invention. That same oligonucleotide could also be 5 used, for example, in a PCR method as a primer for polymerization, but as used herein, that oligonucleotide would then be referred to as a "primer". Herein, oligonucleotides or polynucleotides may contain some modified linkages such as a 10 phosphorothioate bond. As used herein, the terms "complementary" or "complementarity" are used in reference to nucleic acids (i.e., a sequence of nucleotides) related by the well-known base-pairing rules that A pairs with T 15 and C pairs with G. For example, the sequence 5'-A G-T-3', is complementary to the sequence 3'-T-C-A-5'. Complementarity can be "partial," in which only some of the nucleic acid bases are matched according to the base pairing rules. On the other hand, there may 20 be "complete" or "total" complementarity between the nucleic acid strands when all of the bases are matched according to base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of 25 hybridization between nucleic acid strands as known well in the art. This is of particular importance in detection methods that depend upon binding between nucleic acids, such as those of the invention. The term "substantially complementary" refers to any 30 probe that can hybridize to either or both strands of the target nucleic acid sequence under conditions of WO 00/49181 PCT/USOO/04243 -40 low stringency as described below or, preferably, in polymerase reaction buffer (Promega, M195A) heated to 95'C and then cooled to room temperature. As used herein, when the nucleic acid probe is referred to as 5 partially or totally complementary to the target nucleic acid, that refers to the 3'-terminal region of the probe (i.e. within about 10 nucleotides of the 3'-terminal nucleotide position). As used herein, the term "hybridization" is 10 used in reference to the pairing of complementary nucleic acid strands. Hybridization and the strength of hybridization (i.e., the strength of the association between nucleic acid strands) is impacted by many factors well known in the art including the 15 degree of complementarity between the nucleic acids, stringency of the conditions involved affected by such conditions as the concentration of salts, the Tm (melting temperature) of the formed hybrid, the presence of other components (e.g., the presence or 20 absence of polyethylene glycol), the molarity of the hybridizing strands and the G:C content of the nucleic acid strands. As used herein, the term "stringency" is used in reference to the conditions of temperature, 25 ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of 30 complementary base sequences. Thus, conditions of "weak" or "low" stringency are often required when it WO 00/49181 PCT/USOO/04243 -41 is desired that nucleic acids which are not completely complementary to one another be hybridized or annealed together. The art knows well that numerous equivalent conditions can be employed to 5 comprise low stringency conditions. As used herein, the term "Tm" is used in reference to the "melting temperature". The melting temperature is the temperature at which 50% of a population of double-stranded nucleic acid molecules 10 becomes dissociated into single strands. The equation for calculating the Tm of nucleic acids is well-known in the art. The Tm of a hybrid nucleic acid is often estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used 15 for calculating Tm for PCR primers: Tm = [(number of A + T) x 2 0 C + (number of G + C) x 4 0 C]. C.R. Newton et al. PCR, 2 Ed., Springer-Verlag (New York: 1997), p. 24. This formula was found to be inaccurate for primers longer that 20 nucleotides. 20 Id. Other more sophisticated computations exist in the art which take structural as well as sequence characteristics into account for the calculation of Tm. A calculated Tm is merely an estimate; the optimum temperature is commonly determined 25 empirically. The term "homology", as used herein, refers to a degree of complementarity. There can be partial homology or complete homology (i.e., identity). A partially complementary sequence that at least 30 partially inhibits a completely complementary WO 00/49181 PCT/USOO/04243 -42 sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous." When used in reference to a double-stranded 5 nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous", as used herein, refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under conditions of low stringency. 10 When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous", as used herein, refers to a probe that can hybridize to (i.e., is the complement of) the single-stranded nucleic acid template sequence under 15 conditions of low stringency. The term "interrogation position", as used herein, refers to the location of a given base of interest within a nucleic acid probe. For example, in the analysis of SNPs, the "interrogation position" 20 in the probe is in the position that would be complementary to the single nucleotide of the target that may be altered from wild type. The analytical output from a method of the invention provides information about a nucleic acid residue of the 25 target nucleic acid that is complementary to an interrogation position of the probe. An interrogation position is within about ten bases of the actual 3'-terminal nucleotide of the nucleic acid probe, although not necessarily at the 3'-terminal 30 nucleotide position. The interrogation position of the target nucleic acid sequence is opposite the WO 00/49181 PCT/USOO/04243 -43 interrogation position of the probe, when the target and probe nucleic acids are hybridized. The term "identifier nucleotide", as used herein, refers to a nucleotide whose presence is to 5 be detected in a process of the invention to identify that a depolymerization reaction has occurred. The particular application of a method of the invention affects which residues are considered an identifier nucleotide. For a method using ATP detection (e.g. 10 luciferase/luciferin or NADH) wherein, during analysis, all nucleotides released in the depolymerization are "converted" to ATP with an enzyme such as NDPK, all nucleotides released are identifier nucleotides. Similarly, for a method 15 using absorbance detection that does not distinguish between nucleotides, all released nucleotides are identifier nucleotides. For a mass spectrometric detection wherein all the released nucleotides are analyzed, all released nucleotides can be identifier 20 nucleotides; alternatively a particular nucleotide (e.g. a nucleotide analog having a distinctive mass) can be detected. For fluorescence detection, a fluorescently-labeled nucleotide is an identifier nucleotide. The nucleotide may be labeled prior to 25 or after release from the nucleic acid. For radiographic detection, a radioactively-labeled nucleotide is an identifier nucleotide. In some cases, the release of identifier nucleotide is deduced by analyzing the remainder of the probe after 30 a depolymerization step of the invention. Such analysis is generally by a determination of the size WO 00/49181 PCT/USOO/04243 -44 or mass of the remaining probe and can be by any of the described analytical methods (e.g. a fluorescent tag on the 5'-terminus of the probe to monitor its molecular weight following capillary 5 electrophoresis). The term "sample", as used herein, is used in its broadest sense. A sample suspected of containing a nucleic acid can comprise a cell, chromosomes isolated from a cell (e.g., a spread of 10 metaphase chromosomes), genomic DNA, RNA, cDNA and the like. The term "detection", as used herein, refers to quantitatively or qualitatively identifying a nucleotide or nucleic acid within a sample. 15 The term "depolymerization", as used herein, refers to the removal of a nucleotide from the 3' end of a nucleic acid. The term "allele", as used herein, refers to an alternative form of a gene and the term 20 "locus," as used herein, refers to a particular place on a nucleic acid molecule. Detailed Description of the Invention A multiplex method of this invention is 25 used to determine the presence or absence of a plurality of predetermined (known) nucleic acid target sequences in a nucleic acid sample. A nucleic acid target is "predetermined" in that its sequence must be known to design a probe that hybridizes with 30 that target. However, it should be noted that a nucleic acid target sequence, as used with respect to WO 00/49181 PCT/USOO/04243 -45 a process of this invention can merely act as a reporter to signal the presence of a different nucleic acid whose presence is desired to be determined. That other nucleic acid of interest does 5 not have to have a predetermined sequence. Furthermore, a process of the invention is useful in determining the identity of a base within a target where only enough of the sequence is known to design a probe that hybridizes to that target with partial 10 complementarity at the 3'-terminal region of the probe. Such a method utilizes an enzyme that can depolymerize the 3'-terminus of an oligonucleotide probe hybridized to the nucleic acid target sequence 15 to release one or more identifier nucleotides whose presence or absence can then be determined as an analytical output that indicates the presence or absence of the target sequence. A nucleic acid target sequence is 20 predetermined in that a nucleic acid probe is provided to be partially or totally complementary to that nucleic acid target sequence. A nucleic acid target sequence is a portion of nucleic acid sample with which the probe hybridizes if that target 25 sequence is present in the sample. A first step of the method is admixing a sample to be assayed with a plurality of nucleic acid probes. The admixing of the first step is typically carried out under low stringency hybridizing 30 conditions to form a hybridization composition. In such a hybridization composition, the 3'-terminal WO 00/49181 PCT/USOO/04243 -46 region of the nucleic acid probes (i) hybridize with partial or total complementarity to a nucleic acid target sequence that may be present in the sample; and (ii) include an identifier nucleotide in the 3' 5 terminal region. Preferably, a nucleic acid probe is designed to not hybridize with itself to form a hairpin structure in such a way as to interfere with hybridization of the 3'-terminal region of the probe 10 to the target nucleic acid. Parameters guiding probe design are well known in the art. The hybridization composition is maintained under hybridizing conditions for a time period sufficient to form a treated sample that may contain 15 a plurality of predetermined nucleic acid target sequences hybridized with their respective nucleic acid probes. In the event that the sample to be assayed does not contain a target sequence to which a probe 20 hybridizes, no hybridization takes place for that probe. When a method of the present invention is used to determine whether a particular target sequence is present or absent in a sample to be assayed, the resulting treated sample may not contain 25 a substrate for the enzymes of the present invention. As a result, a 3' terminal region identifier nucleotide is not released and the analytical output is at or near background levels. The contemplated method is a multiplex 30 assay in which a plurality of probes is utilized to determine whether one or more of a plurality of WO 00/49181 PCT/USOO/04243 -47 predetermined nucleic acid target sequences is present or absent in a sample. A particularly useful area for such multiplex assays is in screening assays where the usual analytical output indicates that the 5 sought-after nucleic acid is absent. In one illustrative embodiment, a nucleic acid sample is screened for the presence of a plurality of predetermined mutant nucleic acid. In this embodiment, the mutants usually are not present 10 and the analytical output is, for example, at about background levels except where a mutation is present. In another embodiment, a plurality of samples is examined for the presence or absence of microbe specific nucleic acid. Here, again, where a 15 population of healthy individuals, animals, or presumably sterile food is sampled, the absence of the sought-after nucleic acid provides an analytical output that is about background levels, and only in the rare instance does a greater than the background 20 output appear. In a multiplexed embodiment of the above process, the sample is admixed with a plurality of different nucleic acid probes, preferably after amplification of the multiple nucleic acid targets as 25 needed. In this embodiment of the invention, the analytical output for a certain result with one of the probes is distinguishable from the analytical output from the opposite result with all of the probes. 30 In preferred embodiments, the ATP produced via NDPK conversion of released nucleotides in the WO 00/49181 PCT/USOO/04243 -48 presence of ADP is detected by a luciferase detection system or an NADH detection system. In still another embodiment of the present invention, the pyrophosphate transferring step and the phosphate 5 transferring step are performed in a single pot reaction. In other preferred embodiments, if increased sensitivity is required, the ATP molecules can be amplified. In a contemplated multiplex embodiment, 10 information about the presence or absence of a plurality of nucleic acid target sequences is determined using a process of the invention on a single nucleic acid sample, by admixing the sample with a plurality of nucleic acid probes for various 15 nucleic acid targets. In a first multiplex embodiment of the invention, the analytical output obtained when at least one of the nucleic acid probes hybridizes with partial complementarity to its target nucleic acid 20 sequence is greater than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences. Preferably, in such an embodiment, the enzyme whose activity is to depolymerize 25 hybridized nucleic acid to release nucleotides exhibits a 3'-+5'-exonuclease activity, depolymerizing hybridized nucleic acids having one or more mismatched bases at the 3'-terminus of the hybridized probe. 30 In a second multiplex embodiment of the invention, the analytical output obtained when at WO 00/49181 PCT/USOO/04243 -49 least one of said nucleic acid probes hybridizes with partial complementarity to its target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with total 5 complementarity to their respective nucleic acid target sequences. Preferably, in such an embodiment, the enzyme whose activity is to depolymerize hybridized nucleic acid to release nucleotides is a template-dependent polymerase. 10 In a third multiplex embodiment of the invention, the analytical output obtained when at least one of said nucleic acid probes hybridizes with total complementarity to its nucleic acid target sequence is greater than the analytical output when 15 all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences. Preferably, in such an embodiment, the enzyme whose activity is to depolymerize hybridized nucleic acid to release nucleotides is a 20 template-dependent polymerase. In a fourth multiplex embodiment of the invention, the analytical output obtained when at least one of said nucleic acid probes hybridizes with total complementarity to its target nucleic acid 25 sequence is less than the analytical output when all of the nucleic acid probes hybridize with partial complementarity.to their respective nucleic acid target sequences. Preferably, in such an embodiment, the enzyme whose activity is to depolymerize 30 hybridized nucleic acid to release nucleotides exhibits a 3'-+5'-exonuclease activity, depolymerizing WO 00/49181 PCT/USOO/04243 -50 hybridized nucleic acids having one or more mismatched bases at the 3'-terminus of the hybridized probe. The treated sample is admixed with a 5 depolymerizing amount of an enzyme whose activity is to release one or more identifier nucleotides from the 3'-terminus of the probe that is hybridized to the nucleic acid target to form a depolymerization reaction mixture. The choice of enzyme used in the 10 process determines if a match or mismatch at the 3' terminal nucleotide results in release of that 3' terminal nucleotide. Further information regarding specific enzyme reaction conditions is discussed in detail hereinafter. 15 The depolymerization reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom to form a treated 20 reaction mixture. The presence or absence of released identifier nucleotides is then determined to obtain an analytical output. The analytical output indicates the presence or absence of a nucleic acid 25 target sequence in the sample. Processes of the invention can also be concerned with the degree of hybridization of the target to the 3'-terminal region of the probe. Examples hereinafter show that the distinction 30 between a matched and mismatched base becomes less notable as a single mismatch is at a position further WO 00/49181 PCT/USOO/04243 -51 upstream from the 3'-terminal region position. There is very little discrimination between a match and mismatch when a single mismatch is ten to twelve residues from the 3'-terminal nucleotide position, 5 whereas great discrimination is observed when a single mismatch is at the 3'-terminus. Therefore, when the degree of complementarity (partial or total complementarity) of a nucleic acid probe hybridized to a target nucleic acid sequence is referred to 10 herein in regard to an identifier nucleotide, this is to be understood to be referring to within the 3' terminal region, up to about ten residues of the 3' terminal position. In particular embodiments of the invention, 15 it is desirable to include a destabilizing mismatch in or near the 3'-terminal region of the probe. In an example of such an embodiment, the goal is to determine whether a nucleotide at an interrogation position is a match or a mismatch with the target. 20 Better discrimination between match and mismatch at the interrogation position is observed when an intentional mismatch is introduced about 2 to about 10 nucleotides from the interrogation position or preferably about 2 to about 6 nucleotides from the 25 interrogation position. The distinction of the analytical output between matched and mismatched nucleotides when there is more than a single base that is mismatched within the 3'-terminal region can be evident even if 30 mismatches are beyond position 10 from the terminus, for example at position 11 and 12 upstream of the 3'- WO 00/49181 PCT/USOO/04243 -52 terminal nucleotide. Thus, the phrases "about 10" and "3'-terminal region" are used above. The 3' terminal region therefore comprises the approximately 10 residues from the 3'-terminal nucleotide (or 3' 5 terminus) position of a nucleic acid. Hybridization conditions can be empirically ascertained for a control sample for various time periods, pH values, temperatures, nucleic acid probe/target combinations and the like. Exemplary 10 maintenance times and conditions are provided in the specific examples hereinafter and typically reflect low stringency hybridization conditions. In practice, once a suitable set of hybridization conditions and maintenance time periods are known for 15 a given set of probes, an assay using those conditions provides the correct result if the nucleic acid target sequence is present. Typical maintenance times are about 5 to about 60 minutes. In one contemplated embodiment of the 20 invention, the enzyme whose activity is to depolymerize hybridized nucleic acid to release nucleotides from the probe 3'-terminal end is a template-dependent polymerase. In such an embodiment, the reverse of a polymerase reaction is 25 used to depolymerize a nucleic acid probe, and the identifier nucleotide is released when the 3' terminal nucleotide of the nucleic acid probe hybridizes with total complementarity to its nucleic acid target sequence. A signal confirms the presence 30 of a nucleic acid target sequence that has the sequence sufficiently complementary to the nucleic WO 00/49181 PCT/USOO/04243 -53 acid probe to be detected by the process of the invention. In an embodiment that uses a 3'-+5' exonuclease activity of a polymerase, such as Klenow 5 or T4 DNA polymerase (but not limited to those two enzymes), to depolymerize a nucleic acid probe, an identifier nucleotide is released when the 3' terminal residue of the nucleic acid probe is mismatched and therefore there is only partial 10 complementarity of the 3'-terminus of the nucleic acid probe to its nucleic acid target sequence. In this embodiment, to minimize background, the hybrid is typically purified from the un-annealed nucleic acid prior to the enzyme reaction, which releases 15 identifier nucleotides. A signal confirms the presence of a nucleic acid target sequence that is not totally complementary to the nucleic acid probe. In an embodiment that uses a 3'-+5' exonuclease activity of Exonuclease III to 20 depolymerize a nucleic acid probe, an identifier nucleotide is released when the 3'-terminal residue of the nucleic acid probe is matched to the target nucleic acid. A signal confirms the presence of a nucleic acid target that is complementary at the 25 released identifier nucleotide. It is thus seen that hybridization and depolymerization can lead to the release of an identifier nucleotide or to little or no release of such a nucleotide, depending upon whether the 30 probe:target hybrid is matched or mismatched at the 3'-terminal region. This is also dependent on the WO 00/49181 PCT/USO0/04243 -54 type of enzyme used and the type of end, matched or mismatched, that the enzyme requires for depolymerization activity. The magnitude of a contemplated analytical 5 output under defined conditions is dependent upon the amount of released nucleotides. Where an identifier nucleotide is released, an analytical output can be provided that has a value greater than background. Where an identifier nucleotide is not released either 10 because the target sequence was not present in the original sample or because the probe and depolymerizing enzyme chosen do not provide release of a 3'-terminal nucleotide when the target is present, or if the match/mismatch state of the 3' 15 terminal nucleotide did not match that required for the enzyme used to release a 3'-terminal nucleotide, the analytical output is substantially at a background level Depolymerization reactions and enzymes 20 useful in such reactions are discussed in more detail in the parental applications recited hereinbefore and incorporated herein by reference. Template-dependent nucleic acid polymerases capable of pyrophosphorolysis include, but are not 25 limited to, DNA polymerase x, DNA polymerase P, T4 DNA polymerase, Taq polymerase, The polymerase, Tne triple mutant polymerase, Tth polymerase, Tvu polymerase, Ath polymerase, Bst polymerase, E. coli DNA polymerase I, Klenow fragment, Klenow exo minus 30 (exo-), AMV reverse transcriptase, RNA polymerase and MMLV reverse transcriptase. Most preferably, Klenow WO 00/49181 PCT/USOO/04243 -55 exo minus (Klenow exo-) or The triple mutant polymerase is utilized for DNA pyrophosphorolysis reactions because of their efficient utilization of 5' overhanging DNA ends. 5 In a preferred embodiment in the case of the reverse of polymerase activity (pyrophosphorolysis), a preferred substrate is a DNA probe hybridized to a nucleic acid target sequence with total complementarity at its 3'-terminus, 10 including an identifier residue at the 3'-terminal region. A depolymerization reaction can be catalyzed by bacteriophage T4 polymerase in the absence of NTPs depolymerizes a mismatched hybrid. 15 In preferred embodiments, the released nucleotides, XMPs, are produced by nuclease digestion. Nuclease digestion can be accomplished by a variety of nucleases that release a nucleotide with a 5' phosphate, including S1 nuclease, nuclease BAL 31, 20 mung bean nuclease, exonuclease III and ribonuclease H. Nuclease digestion conditions and buffers are known in the art. Nucleases and buffers for their use are available from commercial sources. In an embodiment of the invention where the 25 enzyme's activity is a 3'-+5' exonuclease activity, the hybridized nucleic acid probe is depolymerized from its 3'-terminal nucleotide. In a preferred embodiment in the case of a 3'-+5' exonuclease activity of a polymerase, the preferred substrate is 30 a nucleic acid probe hybridized to a nucleic acid target sequence with partial complementarity at its WO 00/49181 PCT/USOO/04243 -56 3'-terminal region, most preferably with a mismatch at its 3'-terminal residue that is an identifier nucleotide. Preferred reaction mixtures for 5 depolymerization, including suitable buffers for each enzyme, are described in greater detail in the parent application and the Examples. Typically, under these conditions, sufficient NTP or dNTP is released to accurately detect or assay extremely low amounts of 10 nucleic acids (e.g., about 5-1000 picograms). In some preferred embodiments, oligonucleotide probes are typically utilized at about 100 ng to about 1 ptg per 20 iL depolymerization reaction. That amount provides a probe to target 15 weight ratio of about 200:1 to about 1,000:1. In a preferred embodiment of the present invention, nucleic acid polymerase and pyrophosphate (PPj) or an analogue thereof, are added to a hybridized sample containing from less than about 100 20 pg of target nucleic acid, to less than about 10 pg of nucleic acid. Typical target nucleic acids are present at about 1 to about 5 ng in the sample to be assayed, with a target nucleic acid length of about 30 to about 1000 bp being preferred. 25 A depolymerizing enzyme is preferably present in an amount sufficient to depolymerize a hybridized target:probe. That amount can vary with the enzyme used, the depolymerization temperature, the buffer, and the like, as are well-known in the 30 art. For a typical reaction carried out in a 20 pAL volume, about 0.25 to about 1 unit (U) of an enzyme WO 00/49181 PCT/USO0/04243 -57 such as Klenow exo- is used. About 1 to about 5 U of the thermostable enzymes are used for depolymerization at elevated temperatures. Other conditions affecting the 5 depolymerization reactions are discussed in the parent applications cited hereinabove, which are incorporated by reference. Analytical Output 10 The analytical output is obtained by detection of the released identifier products, either the released nucleotides or the remainder of the probe. Exemplary detection systems include the light emitting luciferase detection system, the NADH light 15 adsorption detection system (NADH detection system), fluorescence emissions and mass spectrometry. These detection systems are discussed hereinbelow. The fact that nucleotides were released (a qualitative determination), or even the number of 20 nucleotides released (a quantitative determination) can be deduced through examination of the probe after depolymerization. The determination of the size of an oligonucleotide is well known in the art. For example gel separation and chromatographic 25 separations are well known. Gel imaging techniques that take advantage of fluorescence and absorbance spectroscopy as well as radiographic methods. Mass spectrometry of oligonucleotides is also becoming more common. 30 As is illustrated in the Examples that follow, it can be beneficial to carry out a WO 00/49181 PCT/USOO/04243 -58 contemplated method at elevated temperatures, e.g., about 50 0 C to about 90 0 C. The Tne triple mutant DNA polymerase is described in detail in WO 96/41014, whose disclosures are incorporated by reference, and 5 its 610 residue amino acid sequence is provided as SEQ ID NO:35 of that document. That enzyme is referred to in WO 96/41014 as Tne M284 (D323A,D389A). Briefly, that enzyme is a triple mutant of the polymerase encoded by the thermophilic 10 eubacterium Thermotoga neapolitana (ATCC 49049). The amino-terminal 283 residues of the native sequence are deleted and the aspartic acid residues at positions 323 and 389 of the native sequence are replaced by alanine residues in this recombinant 15 enzyme. This recombinant enzyme is thus a deletion and replacement mutant of the native enzyme. Deletion of the amino-terminal sequence removes the 5' exonuclease activity of the native enzyme, whereas replacement of the two aspartic acid 20 residues removes a magnesium binding site whose presence facilitates exonuclease activity, and this triple mutant also exhibited no 3' exonuclease activity relative to the recombinant native enzyme. This triple mutant enzyme exhibited a half-life at 25 97.5'C of 66 minutes as compared to the full length recombinant enzyme that exhibited a half-life of only 5 minutes at that temperature. A. Detection Of ATP 30 Luciferase detection systems are particularly useful for detecting ATP. In the WO 00/49181 PCT/USOO/04243 -59 presence of ATP and oxygen, luciferase catalyzes the oxidation of luciferin, producing light that can then be quantified using a luminometer. Additional products of the reaction are AMP, pyrophosphate and 5 oxyluciferin. In particularly preferred embodiments, ATP detection buffer referred to as L/L reagent (Promega, FF2021) is utilized. Preferably, about 5 to 10 ng of luciferase are used in the reaction. Although it is 10 not intended that the present invention be limited to a specific concentration of luciferase, greater amounts of luciferase have a tendency to increase non-specific background. It is contemplated that in some 15 embodiments, the dNTPs or NTPs produced by pyrophosphorolysis or nuclease digestion are converted to XTP, which can then be used directly as substrate for luciferase, permitting detection of the nucleic acid. However, the preferred substrate for 20 luciferase is ATP, as demonstrated by Moyer and Henderson, Anal. Biochem., 131:187-89 (1983). When DNA is the initial substrate, NDPK is conveniently utilized to catalyze the conversion of dNTPs to ATP by the following general reaction: 25 Reaction 4: dNTP* + ADP -+ dNDP + ATP* wherein dNTP is a mixture of deoxyribonucleoside triphosphates and dNDP is the corresponding deoxyribonucleoside diphosphate. In Reaction 4, the terminal 5'-triphosphate (P*) of the dNTP is 30 transferred to ADP to form ATP.

WO 00/49181 PCT/USOO/04243 -60 Enzymes catalyzing this reaction are generally known as nucleoside diphosphate kinases (NDPKs). NDPKs are ubiquitous, relatively nonspecific enzymes. For a review of NDPK, see Parks 5 and Agarwal, in The Enzymes, Volume 8, P. Boyer Ed. (1973). The conversion of NTPs or dNTPs to ATP by NDPK is preferably accomplished by adding NDPK and a molar excess of ADP over the amounts of NTPs or dNTPs 10 expected to be produced by pyrophosphorolysis or nuclease digestion, followed by pyrophosphorylation by PRPP synthetase. The utilization of ADP requires optimization of the amount of ADP added. Too much ADP results in high background levels. 15 NDPK (EC 2.7.4.6) preparations from several biological sources are commercially available from several suppliers. For example yeast NDPK is available from Sigma Chemical Co., St. Louis, MO, whereas bovine NDPK is available from ICN 20 Biochemicals, Inc., Costa Mesa, CA. The particular NDPK selected for most uses described herein is typically a matter of choice. Although yeast, bovine or another NDPK can be used in these reactions, it is preferred to utilize a thermostable NDPK such as the 25 Pfu NDPK along with a thermostable depolymerizing enzyme such as the Tne triple mutant DNA polymerase (discussed below), Bst DNA polymerase, Ath DNA polymerase, Taq DNA polymerase and Tvu DNA polymerase along with a reaction temperature of about 50 0 C to 30 about 90 0 C. The use of these thermostable enzymes at an above temperature can enhance the sensitivity of WO 00/49181 PCT/USOO/04243 -61 the method. The The triple mutant DNA polymerase is described in detail in WO 96/41014, whose disclosures are incorporated by reference, and its 610 residue amino acid sequence is provided as SEQ ID NO:35 of 5 that document. That enzyme is referred to in WO 96/41014 as Tne M284 (D323A,D389A). B. Mass Spectrometric Analysis In one method of the invention, the 10 presence of released nucleotides is analyzed via mass spectrometry. In an embodiment of a method using mass spectrometry, the treated reaction mixture is ionized in a manner such that all components of the treated reaction mixture in the molecular weight 15 range of the released identifier nucleotides are measured. Very small differences in molecular weight can be detected using mass spectrographic methods (different isotopes of the same atom are detectable), so any variation from a natural nucleic acid, 20 including a single atom substitution (e.g. a fluorine in place of a hydrogen atom or a replacement of a hydrogen by a deuterium atom) in the identifier nucleotide gives rise to a detectable difference. Nucleic acid analogs used in methods of the invention 25 should not interfere with either the hybridization of the nucleic acid probe or depolymerization of the hybridized probe. Additionally, mass spectrometry can discriminate between individual nucleotides or 30 nucleosides. For example, if the 3'-identifier nucleotide used in the instant invention was a G WO 00/49181 PCT/USOO/04243 -62 nucleoctide, mass spectrometry can be used to detect the release of that G nucleotide in a method of the present invention. Similarly, mass spectrometry can detect the release of an A, T or C nucleotide, based 5 on the differences in atomic weight of these compounds. Thus, in a multiplexing embodiment of the present invention, mass spectrometry can be used to resolve the presence of one or more of these 3' identifier nucleotides. 10 In a particularly useful aspect of this embodiment, a mass spectral technique referred to as DIOS (desorption/ionization on silicon) was recently reported by Wei et al., Nature, 399:243(1999) that can accurately perform one or multiple assays on 15 picogram or attagram amounts using commercially available mass spectrographs adapted with a specialized porous silicon sample well. The older, well known, MALDI mass spectrographic assay techniques can also be utilized. 20 In an embodiment of a multiplex method using mass spectrometry, multiple different identifier nucleotides can be used in the various nucleic acid probes. Using such a technique the presence of the different identifier nucleotides is 25 direct evidence of the presence of the nucleic acid target sequences. C. Fluorescence Spectroscopic Analysis A wide variety of fluorescence detection 30 methods can be used herein. In one exemplary contemplated method, an identifier nucleotide WO 00/49181 PCT/USOO/04243 -63 includes a fluorescent label. An identifier nucleotide can be fluorescently labeled prior to, or after, release of the identifier nucleotide. In an alternative embodiment when the nucleotide is 5 fluorescently labeled, the analytical output is obtained by mass spectrometry. In a preferred embodiment of the invention, the fluorescent label is part of a fluorescent analog of a nucleotide. Fluorescent nucleotide analogs are 10 widely known and commercially available from several sources. An exemplary source is NEN" Life Science Products (Boston, Massachusetts), who offer dideoxy-, deoxy-, and ribonucleotide analogs a labeled with fluorescein, coumarin, tetramethylrhodamine, 15 naphthofluorescein, pyrene, Texas Red*, and Lissamine

M

. Other suppliers include Amersham Pharmacia Biotech (Uppsala, Sweden; Piscataway, New Jersey) and MBI Fermentas, Inc. (Amherst, New York). An advantage to using fluorescent labels 20 and fluorescence spectroscopy analysis is that there are multiple different labels. Such different labels would be particularly useful in a multiplex embodiment of the invention. Different fluorescent labels would be used in different probes, so that the 25 detection of a particular fluorescently-labeled nucleotide analog as a released identifier nucleotide could be used to deduce which nucleic acid targets are present. For example, fluorescein has a 488 nm 30 excitation and 520 nm emission wavelength, whereas rhodamine (in the form of tetramethyl rhodamine) has WO 00/49181 PCT/USOO/04243 -64 550 nm excitation and 575 nm emission wavelength. A fluorescence detector provides an excitation source and an emission detector. The emission wavelengths of 520 nm and 575 nm are easily distinguishable using 5 fluorescence spectroscopy. On a per molecule basis, fluorescence spectroscopy is about 10-fold more sensitive than absorbance spectroscopy. A very wide variety of fluorescence spectroscopy-based detectors are 10 commercially available for reading fluorescence values of single tubes, flow cells and multi-well plates, among others. For example, Labsystems Multiskan models of microplate readers are widely available with a spectral range of 400 to 750 nm, and 15 filters for 340, 405, 414, 450, 492, 540, 620, and 690 nm (e.g. Fisher Scientific, Pittsburgh, Pennsylvania). It is contemplated that a released identifier nucleotide could be labeled before or 20 after depolymerization using cross-linking chemistry well known in the art with commercially available reagents. For example, fluorescein isothiocyanate and rhodamine B isothiocyanate are both available from Aldrich Chemical Company (Milwaukee, Wisconsin). 25 References to fluorescein isothiocyanate's use in labeling biological molecules include Nature, 193:167 (1962), Methods Enzymol. 26:28 (1972), Anal. Biochem., 57:227 (1974), Proc. Natl. Acad. Sci., U.S., 72:459 (1975). 30 It is contemplated that for many embodiments of the invention, it is useful to WO 00/49181 PCT/USOO/04243 -65 separate released fluorescent identifier nucleotides from those bound to an oligonucleotide, such as a probe. Thus, the separation techniques well known in the art and discussed above are useful with such an 5 embodiment, including HPLC fitted with a fluorescence detector. The enhanced sensitivity of fluorescence relative to other spectroscopic techniques can be used to increase the sensitivity of a detection or quantification process of the invention. 10 In the NADH detection system, a combination of two enzymes, phosphoglycerate kinase and glyceraldehyde phosphate dehydrogenase, is used to catalyze the formation of NAD from NADH in the presence of ATP. Because NADH is fluorescent whereas 15 NAD is not, ATP is measured as a loss in fluorescence intensity. Examples of NADH based ATP assays are disclosed in United States Patent Nos. 4,735,897, 4,595,655, 4,446,231 and 4,743,561, and UK Patent Application GB 2,055,200, all of which are herein 20 incorporated by reference. D. Absorbance Spectroscopic Analysis An absorbance spectrographic analysis step is contemplated to provide an analytical output, 25 thereby provide for the determination of the presence or absence released identifier nucleotide, and indicate the presence or absence of said nucleic acid target sequence. This embodiment contemplates the chromatographic separation of a reaction mixture that 30 has been treated with a depolymerizing amount of an enzyme whose activity is to release one or more WO 00/49181 PCT/USOO/04243 -66 nucleotides from the 3'-terminus of a hybridized nucleic acid. In an illustrative embodiment, a multiplexed assay for the presence of several 5 different nucleic acid target sequences in a sample is analyzed by absorbance spectroscopy. Several labeled probes to various nucleic acid target sequences are added to a nucleic acid sample. The labels on the probes may be various nucleotide 10 analogs, a different one for each probe. A depolymerizing enzyme is added, such as Klenow exo-, releasing the labeled nucleotides and other nucleotides from the 3'-termini of probes hybridized to target sequences when the 3' terminal nucleotide 15 is matched. The reaction solution is loaded onto a pre equilibrated High Pressure Liquid Chromatography (HPLC) column and eluted under conditions that separate the nucleotide analogs from the natural 20 nucleotides. Useful media for chromatographic separation of nucleotides, bases, and nucleosides include reverse phase media, such as a reverse phase C18 column or ODS-80TM or ODS-120T TSK-GEL by TosoHaas (Montgomeryville, Pennsylvania), anion exchange 25 media, such as DEAE-25SW or SP-25W TSK-GEL by TosoHaas (Montgomeryville, Pennsylvania), or affinity media, such as Boronate-5PW TSK-GEL by TosoHaas (Montgomeryville, Pennsylvania). Example 5 illustrates an embodiment of the present invention 30 using HPLC.

WO 00/49181 PCT/USOO/04243 -67 The HPLC column is fitted with an absorbance detector to monitor the column effluent. Hence, "absorbance spectroscopy" for this type of analysis. Typical wavelengths for monitoring HPLC 5 detection of nucleotides are 250 nm, 260 nm and 280 nm. Such separations of nucleotides and nucleotide analogs are well known in the art. Revich et al., J. Chromatography, 317:283-300 (1984), and Perrone & Brown, J. Chromatography, 317:301-310 (1984) provide 10 examples of the HPLC separation of dNTPs. Identification of the separated nucleotide analogs can be accomplished by comparison of the retention times (as monitored by absorbance of effluent at various times) of standards of the 15 nucleotide analogs separated on the same HPLC column under the same conditions. Alternatively, the identity of the nucleotide analogs collected in separate fractions (as determined by continually monitoring the absorbance of the column effluent) can 20 be determined by other standard analytical methods, such as nuclear magnetic resonance or atomic analysis (H,C,N). In this illustrative example using depolymerization with Klenow exo-, the presence of a 25 released identifier nucleotide from a particular probe indicates the presence of the target sequence that hybridize with that probe. In an alternative embodiment, the released nucleotides from a depolymerization reaction mixture 30 are separated on a gas chromatograph fitted with an absorbance detector to monitor column effluent.

WO 00/49181 PCT/USOO/04243 -68 Probe-Mediated Specific Nucleic Acid Detection Depolymerization reactions can be used to interrogate the identity of a specific base in a 5 nucleic acid. For example, the identity of single base point mutations, deletions, or insertions in a nucleic acid can be determined as follows. In one embodiment, each nucleic acid probe synthesized is substantially complementary to a 10 target nucleic acid containing or suspected of containing a point mutation. It will be recognized that various hybridization conditions can be used, so as to vary the stringency at which hybridization occurs. Thus, depending upon the system utilized, 15 the complementarity of the probe can be varied. Depending on the length of the probe, the GC content, and the stringency of the hybridization conditions, the probe can have as many as 10 base mismatches with the target nucleic acid, and preferably less than 5 20 mismatches. Most preferably, the probe has only one base mismatch with the target nucleic acid or is completely complementary to the target nucleic acid. A nucleic acid probe comprises single stranded nucleic acid (e.g., DNA or RNA). A probe 25 can be of varying lengths, preferably from about 10 to 100 bases, most preferably about 10 to 30 bases. In particularly preferred embodiments, a probe is complementary to the target at all bases between an interrogation position and 3' end of the nucleic acid 30 probe.

WO 00/49181 PCT/USOO/04243 -69 In preferred embodiments, a probe is designed to have a predetermined nucleotide at an interrogation position. When a complementary probe base pairs or hybridizes to a target nucleic acid, 5 the base at an interrogation position aligns with the base in the nucleic acid target whose identity is to be determined under conditions such that base pairing can occur. It is contemplated that an interrogation position can be varied within the probe. For 10 example, in some preferred embodiments, an interrogation position is preferably within 10 bases of the 3' end of the nucleic acid probe. In still other preferred embodiments, an interrogation position is within 6 bases of the 3' end of the 15 nucleic acid probe. In particularly preferred embodiments, an interrogation position is at the next to last or last base at the 3' end of the nucleic acid probe. In an interrogation embodiment wherein the 20 identity of a base at the interrogation position is desired, four different probes, preferably of equal length, are synthesized, each having a different nucleotide at an interrogation position. Accordingly, it is contemplated that in some 25 embodiments, a set of DNA probes includes a first probe with a deoxyadenosine residue at an interrogation position, a second probe with a deoxythymidine residue at an interrogation position, a third probe with a deoxyguanosine residue at an 30 interrogation position, and a fourth probe with a deoxycytosine residue at an interrogation position.

WO 00/49181 PCT/USOO/04243 -70 Likewise, it is also contemplated that a set of RNA probes includes a first probe with an adenosine residue at an interrogation position, a second probe with a uridine residue at an interrogation position, 5 a third probe with a guanosine residue at an interrogation position, and a fourth probe with a cytosine residue at an interrogation position. In the next step of that interrogation embodiment, the probes are hybridized to the target 10 nucleic acid, if the target nucleic acid is present in the sample, so that a probe nucleic acid-target nucleic acid complex is formed. It is contemplated that hybridization conditions can vary depending on the length and base composition of the probes. In 15 the probe-target nucleic acid complex, the nucleotide at an interrogation position is aligned with the specific base to be identified in the nucleic acid. In the contemplated multiplex embodiment, a set of probes can be used simultaneously. Because the 20 probes differ at an interrogation position, only one of the probes is complementary to the specific base in the target nucleic acid that is aligned with an interrogation position. Preferably, the probes are distinguishable, most preferably the identifier 25 nucleotides are different between the four probes, for example using mass spectrometry or different fluorescent labels. In the next step of that interrogation embodiment, the nucleic acid probe-target nucleic 30 acid complexes are reacted under conditions permitting depolymerization of the probe. The WO 00/49181 PCT/USOO/04243 -71 preferred reaction conditions for depolymerization are described in the parent applications and in the following Examples. The released nucleotides are then detected. 5 In particularly preferred embodiments, the identity of a specific base is determined by comparing the amount of signal produced from each probe. Depolymerization of a hybridized probe proceeds from its 3' end. When the base at an 10 interrogation position is not complementary to the specific base in the nucleic acid, very little or no signal is produced. In yet another preferred embodiment, the probe-mediated specific nucleic acid detection method 15 of the present invention can be used to simply identify or detect a nucleic acid of interest. For this method, nucleic acid probes (e.g., DNA or RNA) are utilized, each of which is substantially complementary to its respective target nucleic acid, 20 which can be RNA or DNA. In a particularly preferred embodiment, each nucleic acid probe is entirely complementary to its target nucleic acid. A nucleic acid probe comprises single-stranded nucleic acid (e.g., DNA or RNA). The probe can be of varying 25 lengths, preferably from about 10 to about 1000 bases, most preferably about 10 to 100 bases. Detection is carried out as described above. The nucleic acid probe-nucleic acid target complex is exposed to conditions permitting 30 depolymerization of the probe, which results in the production of XTPs. Detection of the nucleic acid of WO 00/49181 PCT/USOO/04243 -72 interest is characterized by a difference in the signal generated by the XTPs produced when compared to control sample reactions. For probes completely complementary to their target nucleic acid in the 5 presence of a depolymerizing enzyme which removes matched identifier nucleotides, a signal greater than background would indicate the presence of at least one of the target nucleic acids in the original sample. In a preferred embodiment, each probe 10 contains a distinguishing identifier nucleotide and the detection of an identifier nucleotide released from the target/probe hybrid would determine the presence of a specific target nucleic acid in the original sample. 15 The ability to interrogate the identity of a specific base in a nucleic acid also permits discrimination between nucleic acids from different species, or even from different alleles. The ability to detect and discriminate between nucleic acids of 20 related or unrelated species also permits the identification of species contained within a given nucleic acid-containing sample. For example, the method can be used to determine which species of several related bacteria or virus are contained 25 within a sample (e.g., clinical samples, environmental samples, food samples, or samples from non-human animals). In preferred embodiments of this method, nucleic acids with substantially identical sequences 30 from at least two species or alleles are detected. The region of identity (target nucleic acid sequence) WO 00/49181 PCT/USOO/04243 -73 contains at least a single nucleotide mismatch between the species or alleles in at least one predetermined position and also contains a 3' end and a 5' end or the identification of a nucleic acid 5 sequence unique to each species to be identified. Next, in some embodiments, an RNA or DNA probe that is substantially complementary to the region of identity is synthesized. The probe can be of varying lengths, preferably from about 10 to 1000 10 bases, most preferably about 10 to 100 bases. As above, this complementary probe includes an interrogation position. An interrogation position can be varied within the probe. For example, an interrogation 15 position is preferably within 10 bases of the 3' end of the nucleic acid probe. More preferably, an interrogation position is within 6 bases of the 3' end of the nucleic acid probe. Most preferably, an interrogation position is at the next to last or last 20 base of the 3' end of the nucleic acid probe. The nucleic acid probes are designed so that the base at an interrogation position is complementary to the nucleotide at the predetermined position of one species or allele, but not another 25 due to the mismatch. Likewise, a second probe can be synthesized that is complementary at an interrogation position to the nucleotide at the predetermined position of a second species or allele. A contemplated procedure is employed to 30 identify the presence or absence of multiple species within a given sample. In these embodiments, all WO 00/49181 PCT/USOO/04243 -74 that is required is the identification of substantially identical sequences between species that contain base mismatches or the identification of a nucleic acid sequence unique to each species to be 5 identified. A method contemplated by the present invention has wide applicability in assaying nucleic acids. In some aspects, an endogenous nucleic acid is assayed to determine whether a particular native 10 or mutant sequence is present or absent. This type of analysis is sometimes referred to as genotyping because the genetic makeup of the subject from which the nucleic acid sample is obtained is determined. Speciation, the identity of an organism, such as the 15 identification of a human, dog, chicken, bovine or the like can be determined by use of species-specific nucleic acid probes such as probes to selected regions of the gene encoding cytochrome B. Using a contemplated method, one can 20 illustratively determine whether a human patient, for example, has the Leiden V mutation, a mutant f-globin gene, the cystic fibrosis-related gene in the region of the delta 508 allele, a mutation in a prothrombin gene, congenital adrenal hyperplasia, a translocation 25 that takes place in the region of the bcr gene along with involvement of a segment of the abl gene, the number of repeated sequences in a gene such as are present in THO 1 alleles or the TPOX alleles, as well as the loss of heterozygosity of the locus of certain 30 alleles as is found in certain cancers and also allelic trisomy. Genomic typing can also be used to WO 00/49181 PCT/USOO/04243 -75 assay plant genomes such as that of rice, soy or maize to determine if they contain non-native sequences. The presence or absence in a sample of the genomes of microbes such as Campylobacter jejuni, 5 Listeria, and E. coli 0H157 can be determined, and viral genomes such as that of cytomegalovirus (CMV) or human immunodeficiency virus (HIV) can be analyzed to determine whether a drug-resistant strain is present in a sample. 10 A contemplated method can also be utilized to assay for the presence or absence of nucleic acid that is exogenous to the source of the sample. For example, a contemplated method can be used to assay for the presence of viruses such as hepatitis C virus 15 (HCV), cytomegalovirus (CMV), human immunodeficiency virus (HIV), as well as to determine the viral load in an organism with a disease, such as a human or a plant. A contemplated method can also be used to identify the presence of an exogenous nucleic acid 20 sequence in a plant such as maize, soy or rice. A contemplated method can also be used to assay for the presence of microorganisms such as Listeria monocytogenes, Campylobacter spp., Salmonella spp., Shigella spp. or Escherichia coli (including E. coli 25 E0157) in foodstuffs such as meats, dairy products, and fruit juices. The determination of an appropriate nucleic acid target sequence useful for designing nucleic acid probes for use in a method of the invention is 30 within the skill of the art. Databases of genetic sequences, such as Genbank, can be used to ascertain WO 00/49181 PCT/USOO/04243 -76 the uniqueness of the selected nucleic acid target. Commercially available software for designing PCR primers can be used to assist in the design of probes for use in the invention. 5 In one illustrative embodiment, the predetermined nucleic acid target sequences are associated with blood coagulation, and the nucleic acid probes comprise sequences complementary to nucleic acid sequences associated with blood 10 coagulation. Exemplary sequences associated with blood coagulation comprise (a) a sequence of at least ten nucleotides of the Factor V gene in the region of the Leiden mutation or the corresponding wild type sequence of the Factor V gene; and (b) a sequence of 15 at least ten nucleotides of a prothrombin gene. Preferred exemplary sequences are selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID 20 NO:94, SEQ ID NO:47, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46 and their complementary sequences. FV5 5' CTGCTGCCCTCTGTATTCCTCG 3' SEQ ID NO:14 FV6 5' CTGCTGCCCTCTGTATTCCTTG 3' SEQ ID NO:15 25 PT7 5' GTGACTCTCAGCG 3' SEQ ID NO:87 PT8 5' GTGACTCTCAGCA 3' SEQ ID NO:88 PT9 5' GTGATTCTCAGCG 3' SEQ ID NO:89 PT10 5' GTGATTCTCAGCA 3' SEQ ID NO:90 FV7 5' GACAAAATACCTGTATTCCTCG 3' SEQ ID NO:91 30 FV8 5' GACAAAATACCTGTATTCCTTG 3' SEQ ID NO:92 PT3 5' GGAGCATTGAGGCTCG 3' SEQ ID NO:93 WO 00/49181 PCT/USOO/04243 -77 PT4 5' GGAGCATTGAGGCTTG 3' SEQ ID NO:94 11432 5' GACAAAATACCTGTATTCCTTG 3' SEQ ID NO:47 11265 5' GTGATTCTCAGCA 3' SEQ ID NO:44 11266 5' GTGATTCTCAGCG 3' SEQ ID NO:45 5 9919 5' GACAAAATACCTGTATTCCTCG 3' SEQ ID NO:46 In another contemplated embodiment, the speciation of a nucleic acid sample is determined using a suite of species probes in a single assay. 10 Kits containing probes for such an assay are also contemplated. Exemplary probes shown to be specific for human mitochondrial DNA are SEQ ID NO:50)) and 11583 (SEQ ID NO:51)). An exemplary common probe, 11582 (SEQ ID NO:52), gives a positive reaction with 15 bovine, chicken, dog and human species. An exemplary chicken-specific probe, 11577 (SEQ ID NO:53), is specific for chicken mitochondrial DNA, while another chicken-specific probe, 11584 (SEQ ID NO:54), gives a signal with bovine and human in addition to chicken 20 genomic DNA. An exemplary cow-specific probe, 11588 (SEQ ID NO:55), provides a strong signal with cow target DNA, but also gives a signal with human, chicken and dog nucleic acid. An exemplary dog specific probe, 11586 (SEQ ID NO:56), provides a 25 stronger signal with dog DNA than it does with cow DNA. The sequences complementary to the probes listed below are also contemplated as probe sequences. 30 11576 huzool 5' CCAGACGCCTCA 3' SEQ ID NO:50 11583 huzoo2 5' ACCTTCACGCCA 3' SEQ ID NO:51 WO 00/49181 PCT/USOO/04243 -78 11582 comzoo 5' TGCCGAGACGT 3' SEQ ID NO:52 11577 chzool 5' GCAGACACATCC 3' SEQ ID NO:53 11584 chzoo2 5' GGAATCTCCACG 3' SEQ ID NO:54 11588 cozoo2 5' ACATACACGCAA 3' SEQ ID NO:55 5 11586 dozoo2 5' ATATGCACGCAA 3' SEQ ID NO:56 In a further embodiment, genetic screening for a target sequence in a nucleic acid sample is contemplated using a plurality of probes to the 10 various targets in a single assay. Kits containing probes for such genetic testing are also contemplated. In an exemplary embodiment, the steroid 21-hydroxylase gene is screened for mutations correlated with a group of autosomal recessive 15 diseases collectively known as congenital adrenal hyperplasia (CAH). Exemplary CAH probes for use in a method and kit according to the invention are listed below. The complementary sequences to the probes listed below are also contemplated as probe 20 sequences. 11143 5' CGGAGCCTCCACCTCCCG SEQ ID NO:23 CAH interrogator oligo 6 (wild type) for mutation site 1 25 11085 5' CACCCTCCAGCCCCCAGC 3' SEQ ID NO:24 CAH interrogator oligo 2 (pseudogene/mutant) for mutation site 2 11084 5' CGGAGCCTCCACCTCCTG 3' SEQ ID NO:25 CAH interrogator oligo 1 (pseudogene/mutant) for 30 mutation site 1 11086 5' CCTCACCTGCAGCATCAAC 3' SEQ ID NO:26 WO 00/49181 PCT/USOO/04243 -79 CAH interrogator oligo 3 (pseudogene/mutant) for mutation site 3 11144 5' CACCCTCCAGCCCCCAAC 3' SEQ ID NO:27 CAH interrogator oligo 7 (wild type) for mutation 5 site 2 11145 5' CCTCACCTGCAGCATCATC 3' SEQ ID NO:28 CAH interrogator oligo 8 (wild type) for mutation site 3 11087 5' CCTGGAAGGGCACTT 3' SEQ ID NO:29 10 CAH interrogator oligo 4 (pseudogene/mutant) for mutation site 4 11146 5' CCTGGAAGGGCACGT 3' SEQ ID NO:30 CAH interrogator oligo 9 (wild type) for mutation site 4 15 11088 5' GATTCAGCAGCGACTGTA 3' SEQ ID NO:31 CAH interrogator oligo 5 (pseudogene/mutant) for mutation site 5 11147 5' GATTCAGCAGCGACTGCA 3' SEQ ID NO:32 CAH interrogator oligo 10 (wild type) for mutation 20 site 5 11287 5' CGAGGTGCTGCGCCTGCG 3' SEQ ID NO:33 CAH interrogation oligo 11 (wild type) for mutation site 6 11288 5'CGAGGTGCTGCGCCTGTG 3' SEQ ID NO:34 25 CAH interrogation oligo 12 (pseudogene/mutant) for mutation site 6 11641 5'GGGATCACATCGTGGAGATG 3' SEQ ID NO:35 CAH interrogation oligo 23 (wild type) for mutation site 7 30 11642 5'GGGATCACAACGAGGAGAAG 3' SEQ ID NO:36 WO 00/49181 PCT/USOO/04243 -80 CAH interrogation oligo 24 (pseudogene/mutant) for mutation site 7 Preferably in an assay of genomic or 5 mitochondrial, or otherwise large, complex nucleic acid sample sources using a multiplex method of the invention, a segment of nucleic acid from the genome or mitochondrial nucleic acid is amplified or otherwise isolated to make a sample for conducting a hybridization 10 assay according to the invention. Alternatively, a method of the invention that results in multiple depolymerizable hybrids in the presence of a single target molecule as discussed in the copending application U.S. Serial No. 09/358,972, filed on July 15 21, 1999, is preferred, for example assays forming hairpins (e.g. REAPERTM), or using self-annealing primers. (This application is a continuation-in-part of U.S. patent application Serial No. 09/358,972, the disclosures of which are incorporated herein by 20 reference and published on the internet at http://www.promega.com/pt/pend/09358972.pdf.) In another exemplary multiplex embodiment, predetermined nucleic acid target sequences are associated with cystic fibrosis, and the nucleic acid 25 probes comprise sequences complementary to nucleic acid sequences associated with cystic fibrosis. In one embodiment of this aspect, the target and probes are associated with the cystic fibrosis delta F508 mutation. Particularly preferred exemplary cystic fibrosis 30 associated sequences are SEQ ID NO:95 or SEQ ID NO:96 and their complementary sequences. CF3 5' CATCATAGGAAACACCAAG 3' SEQ ID NO:95 CF4 5' CATCATAGGAAACACCAAT 3' SEQ ID NO:96 35 WO 00/49181 PCT/USO0/04243 -81 Information is currently available and will continue to be made available during the lifetime of this patent as to certain nucleic acid sequences that are associated with various species or diseases. It 5 is contemplated that any of these nucleic acid sequences are useful in a method of the invention and that combinations of probes to the various targets are useful in a multiplex kit according to the present invention. 10 An exemplary cancer-associated nucleic acid sequence discussed in more detail in the parent application, U.S. Serial No. 09/358,972, filed on July 21, 1999, is contemplated for use in a multiplex genetic screening kit and method along with other 15 probes. The nucleic acid probes BA3 (SEQ ID NO:97) and BA4 (SEQ ID NO:98) are useful in detecting a particular translocation that takes place in the region of the bcr gene, and a segment of the abl gene that is involved with the translocation. 20 BA3 5' TGGATTTAAGCAGAGTTCAAGT 3' SEQ ID NO:97 BA4 5' TGGATTTAAGCAGAGTTCAAAA 3' SEQ ID NO:98 Amplification of the Sample Target or a Detection 25 Target A target nucleic acid sequence is typically amplified prior to use of a contemplated method. However, where a sufficient number of repeated nucleotide sequences are present in the native sample 30 as in the human Alu sequence or the E. coli rep WO 00/49181 PCT/USOO/04243 -82 sequence, amplification is often not needed prior to carrying out a contemplated method. Several methods are known in the art to amplify a region of DNA. These include polymerase 5 chain reaction, ligase chain reaction, repair chain reaction, amplification of transcripts, self sustained sequence replication (3SR), ligation activated transcription (LAT), strand displacement amplification (SDA) and rolling circle replication. 10 A claimed process contemplates prior treatment of a nucleic acid sample using any amplification method known in the art at the time of practicing a claimed process to detect the presence of a nucleic acid target in the sample. 15 Multiplex Assays Using Hairpin Structures Although it is preferred that the probes be constructed to be free of hairpin structures, assays in which hairpin structures are constructed are also 20 useful. An embodiment of the invention contemplates use of a hairpin structure for determining the presence or absence of a plurality of nucleic acid target sequences in a nucleic acid sample with a probe that is hybridized to the target and then 25 modified to be able to form a hairpin structure. This embodiment comprises the following steps. A treated sample is provided that contains a nucleic acid sample that may include a plurality of nucleic acid target sequences having an interrogation 30 position. The target sequences, if present in the nucleic acid sample hybridize with their respective WO 00/49181 PCT/USOO/04243 -83 nucleic acid probes. A probe is comprised of at least two sections. The first section contains the probe 3'-terminal about 10 to about 30 nucleotides. These nucleotides are complementary to the target 5 strand sequence at positions beginning about 1 to about 30 nucleotides downstream of the interrogation position. The second section of the probe is located at the 5'-terminal region of the probe and contains about 10 to about 20 nucleotides of the target 10 sequence. This same sequence, therefore, exists in both the target and the probe in the same 5' to 3' orientation. This sequence spans the region in the target from the nucleotide at or just upstream (5') of the interrogation position, to the nucleotide just 15 upstream to where the 3'-terminal nucleotide of the probe anneals to the target. An optional third section of the probe, from zero to about 50, preferably from zero to about 20, nucleotides in length and comprising a sequence that does not 20 hybridize with either the first or second section, is located between the first and second sections of the probe. The hybridized probes of the treated sample are extended in a template-dependent manner, by 25 admixture with dNTPs and a template-dependent polymerase, at least through the interrogation position, thereby forming an extended probe/target hybrid. In a preferred embodiment, the length of the probe extension is limited by omission from the 30 extension reaction of a dNTP complementary to a nucleotide of the target sequence that is present WO 00/49181 PCT/USOO/04243 -84 upstream of the interrogation position and absent between the nucleotide complementary to the 3'-end of the interrogation position. The extended probe/target hybrid is 5 separated from any unreacted dNTPs; i.e., purified at least to the degree needed to use the extended probe strand to determine the presence or absence of the interrogation region in the sample or the identity of the base at the interrogation position. The extended 10 probe/target hybrid is denatured to separate the strands. The extended probe strands are permitted to form hairpin structures. It is preferred that the polymerase enzyme utilized for an extension reaction be a template 15 dependent polymerase that is free of activity that adds a 3'-terminal deoxyadenosine in a template nonspecific manner. Thus, it is preferred to use other than a polymerase such as Taq for a contemplated extension. 20 A treated reaction mixture is formed by admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3'-terminus of an extended probe hairpin 25 structure. The reaction mixture is maintained under depolymerizing conditions for a time period sufficient for the depolymerizing enzyme to release 3'-terminus nucleotides, and then analyzed for the presence of released identifier nucleotides. The 30 analytical output indicates the presence or absence of the nucleic acid target sequence. That analytical WO 00/49181 PCT/USOO/04243 -85 output can be determined as discussed elsewhere herein. A still further embodiment of the invention, such as that termed REAPER m and 5 demonstrated in Example 89 and Figure 2, also contemplates use of hairpin structures in determining the presence or absence of a nucleic acid target sequence, or a specific base within the target sequence, in a nucleic acid sample, and comprises the 10 following steps. A treated sample is provided that contains a nucleic acid sample that may include a plurality of nucleic acid target sequences hybridized with its respective first nucleic acid probe strand (Fig. 2A). 15 The hybrid is termed the first hybrid. The first probes are comprised of at least two sections. The first section contains the probe 3'-terminal about 10 to about 30 nucleotides that are complementary to the target nucleic acid sequence at 20 a position beginning about 5 to about 30 nucleotides downstream of the target interrogation position. The second section of a first probe contains about 5 to about 30 nucleotides that are a repeat of the target sequence from the interrogation position to about 10 25 to about 30 nucleotides downstream of the interrogation position, and does not hybridize to the first section of the probe. That is, the second sequence is a repeat of the region in the target sequence from the interrogation position downstream 30 to the position where the 3'-terminal nucleotide of the first probe aligns with the target. An optional WO 00/49181 PCT/USOO/04243 -86 third section of the probe, located between the first and second sections of the probe, is zero to about 50, preferably to about 20, nucleotides in length and comprises a sequence that does not hybridize to 5 either the first or second section. The first hybrid in the treated sample is extended at the 3'-end of the first probes, thereby extending the first probe past the interrogation position and forming an extended first hybrid (Fig. 10 2B) whose sequence includes an interrogation position. The extended first hybrid is comprised of the original target nucleic acid and extended first probe. The extended first hybrid is then denatured in an aqueous composition to separate the two nucleic 15 acid strands of the hybridized duplex and form an aqueous solution containing separated target nucleic acids and separated extended first probes. Second probes are about 10 to about 2000, more preferably about 10 to about 200, most 20 preferably about 10 to about 30 nucleotides in length and are complementary to their respective extended first probes at a position beginning about 5 to about 2000, preferably about 5 to about 200, nucleotides downstream of the interrogation position in extended 25 first probe, anneal to the extended first probes, thereby forming the second hybrid (Fig. 2C). The second hybrid is extended at the 3'-end of the second probes until that extension reaches the 5'-end of the extended first probes, thereby forming a second 30 extended hybrid (Fig. 2D) whose 3'-region includes an identifier nucleotide.

WO 00/49181 PCT/USOO/04243 -87 An aqueous composition of the extended second hybrid is denatured to separate the two nucleic acid strands; i.e., the extended second probes and the extended first probes. The aqueous 5 composition so formed is cooled to form a "hairpin structure" from the separated extended second probes (Fig. 2E) when the target sequence is present in the original nucleic acid sample. Thus, when the target sequence is present in the original nucleic acid 10 sample, the 3'-terminal sequence of the second extended probes in the second extended hybrid hybridize with the sequence of the second extended probes from a region comprising the interrogation position and nucleotides downstream from the 15 interrogation position of second extended probe to the nucleotide position where the 3'-terminal nucleotide of the original (first-named) probes annealed to the original target. A treated reaction mixture is formed by 20 admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3'-terminus of a nucleic acid hybrid. The reaction mixture is maintained under depolymerizing 25 conditions for a time period sufficient to release 3'-terminal region identifier nucleotides, and then analyzed for the presence of released identifier nucleotides. The analytical output indicates the presence or absence of the various nucleic acid 30 target sequences. Again, the analytical output can be determined by one of the several methods discussed WO 00/49181 PCT/USOO/04243 -88 elsewhere herein. In one embodiment of this multiplex hairpin-forming probe method, the analytical outputs from the various nucleic acid target sequences are distinguishable. 5 As was the case in the previous embodiment, dNTPs are utilized in the extension reactions. It is preferred that the hairpin structures be separated from the dNTPs prior to depolymerization to enhance the analysis for the identifier nucleotide. 10 Kits Other embodiments of the invention contemplate a kit for determining the presence or absence of a plurality of predetermined nucleic acid 15 target sequences in a nucleic acid sample. Such a kit comprises an enzyme whose activity is to release one or more nucleotides from the 3' terminus of a hybridized nucleic acid probe and at least one nucleic acid probe, said nucleic acid probe being 20 complementary to a nucleic acid target sequence. Preferably the enzyme whose activity is to release nucleotides in the kit is a template dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic 25 acids whose bases in the 3'-terminal region are matched with total complementarity. Alternatively, the enzyme whose activity is to release nucleotides in the kit exhibits a 3' to 5' exonuclease activity, depolymerizing hybridized nucleic acids having one or 30 more mismatched bases at the 3' terminus of the hybridized probe.

WO 00/49181 PCT/USOO/04243 -89 In a preferred embodiment, the enzyme capable of catalyzing pyrophosphorolysis is, but is not limited to Taq polymerase, Tne polymerase, Tne triple mutant polymerase, Tth polymerase, Tvu 5 polymerase, Ath polymerase, T4 DNA polymerase, Klenow fragment, Klenow exo minus, E. coli DNA polymerase I, AMV reverse transcriptase, MMLV reverse transcriptase, or poly(A) polymerase. In another preferred embodiment, the kit contains an exonuclease 10 such as S1 nuclease, nuclease BAL 31, mung bean nuclease, exonuclease III and ribonuclease H. Either of the above enzyme types is utilized in a contemplated method in a depolymerizing effective amount. That is, the enzyme is used in an 15 amount that depolymerizes the hybridized probe to release an identifier nucleotide. This amount can vary with the enzyme used and also with the temperature at which depolymerization is carried out. An enzyme of a kit is typically present in an amount 20 that conveniently permits the distribution of about 0.1 to 100 U per reaction; in particularly preferred embodiments, the concentration is about 0.5 U/reaction. An amount of enzyme sufficient to carry out at least one assay, with its controls is 25 provided. In an alternative preferred embodiment, the enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotides is a thermostable polymerase. Preferred thermostable 30 polymerases are the Tne triple mutant DNA polymerase, Klenow exo-, Klenow, T4 DNA polymerase, Ath DNA WO 00/49181 PCT/USOO/04243 -90 polymerase, Taq DNA polymerase and Tvu DNA polymerase, the Tne triple mutant DNA polymerase and Tvu DNA polymerase are particularly preferred. It is to be understood that such a kit is 5 useful for any of the methods of the present invention. The choice of particular components is dependent upon the particular method the kit is designed to carry out. Additional components can be provided for detection of the analytical output, as 10 measured by the release of identifier nucleotide, or by detection of the remaining probe after depolymerization. In one embodiment, a kit has nucleic acid probes that comprise fluorescent labels. In a method 15 using such a kit, either the released identifier nucleotide or the remaining probe is determined using fluorescence spectroscopy, depending on the location of the fluorescent label within the probe. In an alternative embodiment, the released nucleotides are 20 separated from depolymerized probes, and the remaining probe or released nucleotide is fluorescently labeled. Such an embodiment contemplates the provision of fluorescent labels and/or a magnetic nucleic acid separation medium. 25 The above fluorescent embodiments contemplate that the fluorescent labels are distinguishable. In another embodiment, a kit has nucleic acid probes that comprise a non-natural nucleotide analog as an identifier nucleotide. In a 30 contemplated method using such a kit, the WO 00/49181 PCT/USOO/04243 -91 depolymerization is assayed for using mass spectrometry. The kit optionally further comprises instructions for detecting said nucleic acid by 5 depolymerization. The instructions present in such a kit instruct the user on how to use the components of the kit to perform the various methods of the present invention. These instructions can include a description of the detection methods of the 10 invention, including detection by luminescence spectroscopy, mass spectrometry, fluorescence spectroscopy, and absorbance spectroscopy. In one embodiment, the invention contemplates a kit for determining the presence or 15 absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample comprising the following components: an enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotide as a nucleoside triphosphate 20 from hybridized nucleic acid probe; pyrophosphate; and a plurality of nucleic acid probes, wherein the nucleic acid probes are complementary to their respective predetermined nucleic acid target sequence. 25 The enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotide is as described hereinabove. Preferably, the plurality of nucleic acid probes are for related applications for a useful and convenient multiplex 30 assay. For example, the detection of several genetic disease markers (e.g. Factor V Leiden and WO 00/49181 PCT/USOO/04243 -92 prothrombin), a suite of human identity screens, the detection of a series of harmful microorganisms (e.g. certain E. coli strains, camphylobacter jejuni, and salmonella or HIV-I, HIV-II, drug-resistant HIV-I, 5 Hepatitis C and Hepatitis B), or to check a plant for a series of nucleic acids. Several examples of such probes are discussed hereinabove. In a contemplated kit for multiplexed probe-mediated specific nucleic acid detection, the 10 kit contains a plurality of nucleic acid probes for nucleic acid targets of interest. Preferably, where the kits contain multiple probes, each of the probes is designed to interrogate a different target DNA sequence. 15 The invention also contemplates kits containing instructions for use in interrogating the identity of a specific base within a nucleic acid target using a plurality of probes with different bases at the interrogation position and 20 distinguishable probes. The invention also contemplates kits containing instructions for use in simultaneously discriminating between two homologous nucleic acid targets that differ by one or more base pairs by providing a distinguishable probe for each 25 target. Alternatively, the invention contemplates a kit containing instructions for use in the simultaneous discrimination between a suite of nucleic acid targets that differ from a homologous nucleic acid target by one or more base pairs using 30 distinguishable probes. The invention further contemplates a kit containing instructions for use in WO 00/49181 PCT/USOO/04243 -93 determining whether a sample contains a plurality of nucleic acid targets having a deletion or insertion mutation. The types of nucleic acid probes that can be included in the kits and their uses are described 5 in greater detail below. Example 1: Multiplex Analysis of Alleles at One Interrogation Site For a wide variety of genetic disorders, 10 only a very small percentage of samples will have a particular single nucleotide polymorphism (SNP) at any one site. For this reason, it can be much more efficient in these cases to screen for the presence of groups of mutant alleles and to perform secondary, 15 single probe tests only if there is a positive signal for any of the probes designed to detect the mutant sites. Such a form of multiplex analysis will be performed in this example. Multiple probes designed to detect a mutant 20 form of a gene in the CMV genome are used in one reaction and the signal from this reaction is compared to that from a probe that is specific for the non-mutated sequence. In this example, the SNP sites are separated by only one base and the alleles 25 are provided as pure nucleic acid target species. Oligonucleotides CV19 (SEQ ID NO:1) and CV20 (SEQ ID NO:2) encode a segment of the CMV genome around position 1784 of the viral genome and these probes encode the non-mutant form of a gene. 30 Oligonucleotides CV21 (SEQ ID NO:3) and CV22 (SEQ ID NO:4) encode the same genome segment as CV19 and CV20 WO 00/49181 PCT/USOO/04243 -94 but encode a form of the gene where a Leu codon in the encoded protein is altered to encode a Ser codon. Oligonucleotides CV23 (SEQ ID NO:5) and CV24 (SEQ ID NO:6) also encode the same genome 5 segment as CV19 and CV20, but these oligonucleotides encode a form of the genome where the same Leu codon mutated in CV21 and CV22 is altered to a Phe codon. These oligonucleotides are used here as target nucleic acids for interrogation in this example. 10 Oligonucleotide probe CV25 (SEQ ID NO:7) exactly matches a region of CV19 and is designed to detect the non-mutated form of the gene. Oligonucleotide probe CV26 (SEQ ID NO:8) exactly matches a segment within CV21 and is designed to 15 detect the version of the gene where the Leu codon has been mutated to a Ser codon. Oligonucleotide probe CV27 (SEQ ID NO:9) exactly matches a segment within CV24 and is designed to detect the version of the target where the Leu codon has been mutated to a 20 Phe codon. The target nucleic acid pairs CV19 and CV20, CV21 and CV22, and CV23 and CV24 were dissolved at 1 mg/mL in water, annealed by heating the solutions 95 0 C for 5 minutes and permitting to cool at 25 room temperature for 10 minutes. Subsequently, the solutions were diluted to 3.3 ptg/mL with water. The probes CV25, CV26 and CV27 were dissolved at 1 mg/mL in water. 30 The following solutions were assembled.

WO 00/49181 PCT/USOO/04243 -95 Solution CV CV CV (19+20) (21+22) (23+24) CV25 CV26 CV27 Water #1 and #2 1 pL -- -- 1 PL -- -- 18 pL #3 and #4 1 L -- 1 pL 1 pL 17 pL #5 and #6 -- 1 pL -- 1 pL 18 L #7 and #8 -- 1 L -- -- 1 L 1 pL 17pL #9 and #10 -- -- 1 IL 1 pL -- -- 18 pL #11 and #12 -- -- 1 pL -- 1 pLL 1 pL 17 pL These solutions were heated at 95 0 C for three minutes then cooled at room temperature for 10 minutes. 5 The following master mix was assembled and mixed. Component Volume 1oX DNA Polymerase Buffer 60 ptL (Promega, M195A) 40 mM Sodium Pyrophosphate 7.5 pL (Promega, C350B) Klenow exo- (10U/piL) 7.5 tL (Promega, M218B) NDPK (1U/ptL) 3 tL 10 pM ADP 6 p.L Water 216 tL After solutions 1-12 had cooled at room 10 temperature, 20 [pL of this master mix were added to each solution, and the solutions were heated to 37 0 C for 15 minutes. After this heating step, a 4 pL sample of each solution was added to 100 tL L/L WO 00/49181 PCT/USOO/04243 -96 reagent (Promega, F202A) and the light produced by the resulting reaction was read immediately using a Turner TD 20/20 luminometer. The following results were obtained. 5 Solution samples Relative Light Units #1 115.7 #2 120.9 #3 20.85 #4 20.10 #5 9.99 #6 9.41 #7 102.4 #8 95.2 #9 12.56 #10 12.54 #11 240.3 #12 238.9 The results from the duplicate solutions were averaged and are presented in the table below. 10 WO 00/49181 PCT/USO0/04243 -97 Average Signal from Probe Types Nucleic Wild Type Mutant Probes Ratio* Acid Target Probe Multiplexed Wild Type Target 118.3 20.48 5.78 Leu to Ser Target 9.7 98.8 0.10 Leu to Phe Target 12.6 239.6 0.05 *Ratio is determined by dividing the signal from the wild type probe by the signal from the multiplexed mutant probes. 5 These data show that the use of both mutant probes in one reaction permits either probe to give a signal if the appropriate target is added to the reaction. The signal ratios produced by the probes 10 designed to detect the mutant target when either probe matches the target are significantly different than from when the wild type target is used with the wild type probe. Thus, comparison of the signals as described above permits the user to know that a 15 mutation is present in the tested target at either of the interrogation sites. CV19 5' CTCTTTAAGCACGCCGGCGCGGCCTGCCGCGCGTTGGAGAACGGCAAGCTC 20 ACGCA 3' SEQ ID NO:1 WO 00/49181 PCT/USOO/04243 -98 CV20 5'CAGCAGTGCGTGAGCTTGCCGTTCTCCAACGCGCGGCAGGCCGCGCCGGCG TGCTT 3' SEQ ID NO:2 5 CV21 5'CTCTTTAAGCACGCCGGCGCGGCCTGCCGCGCGTCGGAGAACGGCAAGCTC ACGCA 3' SEQ ID NO:3 CV22 10 5'CAGCAGTGCGTGAGCTTGCCGTTCTCCGCGCGCGGCAGGCCGCGCCGGCGT GCTT 3' SEQ ID NO:4 CV23 5'CTCTTTAAGCACGCCGGCGCGGCCTGCCGCGCGTTTGAGAACGGCAAGCTC 15 ACGCA 3' SEQ ID NO:5 CV24 5'CAGCAGTGCGTGAGCTTGCCGTTCTCAAACGCGCGGCAGGCCGCGCCGGCG TGCTT 3' SEQ ID NO:6 20 CV25 5' GGCGCGGCCTGCCGCGCGTTG 3' SEQ ID NO:7 CV26 5' GGCGCGGCCTGCCGCGCGTCG 3' SEQ ID NO:8 25 CV27 5' GCGTGAGCTTGCCGTTCTCCG 3' SEQ ID NO:9 Example 2: Multiplexed Genome Analysis on Multiple Templates For a wide variety of genetic disorders, 30 only a very small percentage of samples exhibit a particular single nucleotide polymorphism at any one WO 00/49181 PCT/USOO/04243 -99 site. For this reason, it can be more efficient in these cases to screen for the presence of groups of mutant alleles and to perform secondary, single probe tests only if there is a positive signal for any of 5 the probes designed to detect the mutant sites. Such a form of multiplex analysis will be performed in this example. Multiple probes designed to detect a mutant form of two different target genes are used in one 10 reaction, and the signal from this reaction is compared to that from a probe that is specific for one of the non-mutated sequences. Thus, in this example, multiple SNP sites on multiple targets are interrogated in one reaction. 15 The targets and probes used in this study are: FV(1+2) (SEQ ID NO:10 and SEQ ID NO:11, respectively) FV(3+4) (SEQ ID NO:12 and SEQ ID NO:13, respectively), FV5 (SEQ ID NO:14), FV6 (SEQ ID NO:15), 9162 (SEQ ID NO:16), 9165 (SEQ ID NO:17), 20 9163 (SEQ ID NO:18), 9166 (SEQ ID NO:19), and CV2 (SEQ ID NO:20). A synthetic first nucleic acid target of the Factor V gene was designed to have the wild type sequence that contains a G at position 32 of FV1 (SEQ ID NO:10). The complementary strand, 25 FV2, (SEQ ID NO:11) has 4 additional bases at its 3' terminus. A second synthetic nucleic acid target of Factor V was designed to have the Leiden mutation, an A residue at position 32 of FV3. The mutant complementary strand, FV4, also had 4 additional 30 bases at its 3' terminus. The nucleic acid target oligonucleotides, FV1 to FV4, were separately WO 00/49181 PCT/US00/04243 -100 dissolved at a concentration of one mg/mL in water. Oligonucleotides 9162 and 9163 are complementary and have a segment of the wild type CMV genome. Oligonucleotides 9163 and 9166 are complementary and 5 have the same segment of the viral genome, but they contain a single base change present in a known drug resistant form of the virus. Equal volumes of one mg/mL 9162 and 9165 were combined to serve as wild type target for CMV. Equal volumes of one mg/mL 9163 10 and 9166 were combined to serve as the mutant target for CMV. Oligonucleotide CV2 represents an oligonucleotide designed to detect the drug resistant form of the CMV sequence. All the target DNAs [FV(1+2), FV(3+4), 15 9162+9165, 9163+9166] were diluted to 0.3 pg/mL with water. The other oligonucleotides were dissolved to 1 mg/mL with water. These compositions were used to assemble the following solutions. Soln FV5 FV6 CV2 9162 9163 FV (1+2) FV (3+4) Water 9165 9166 1 -- 20 pL 2 -- 1 pL - -- 19 pL 3 -- -- 1 L -- -- 19 pL 4 -- 1 L 1 pL -- -- 18 iL 5 -- 1 iL - -- 19 [IL 6 -- -- 1 tL -- -- 19 tL 7 - -- 1 pL -- 19 [pL 8 -- 1 L 19 pL 9 -- 1 LL -- 1[L -- 18 pL 10 -- 1 iL - 1 pL 18 [L 11 -- -- 1[L 1 PL -- 18 pL WO 00/49181 PCT/USOO/04243 -101 Soln FV5 FV6 CV2 9162 9163 FV(1+2) FV(3+4) Water 9165 9166 12 -- -- 1 [L 1 [pL 18 [L 13 -- -- 20 pL 14 1 pL -- -- 1 pL -- 1 PL -- 17 L 15 1 IL --- 1 PL 1 pL -- 17 IL 16 1 L -- -- .1 pL 1 IL 17 PL 17 1 [L -- 1 [L 1 pL 17 [L 18 -- 1 L 1 pL 1 PL -- 1[pL -- 16 pL 19 -- 1 [L 1 pL -- 1 pL 1 pL -- 16 pL 20 -- 1 [L 1 pL 1 pL -- -- 1 L 16 pL 21 -- 1 [L 1 pL -- 1 pL -- 1 L 16 pL These 21 solutions, in triplicate, were heated to 92 0 C for 11 minutes, then cooled approximately 1 hour at room temperature. 5 The following master mix was assembled and mixed. Component Volume Water 1008 pL 1OX DNA Polymerase Buffer 280 tL (Promega, M195A) Klenow exo- (1U/pLL) 35 tL (Promega, M218B) 40mM Sodium Pyrophosphate 35 ptL (Promega, C350B) 10[LM ADP 28 pL NDPK (1U/pL) 14 pL WO 00/49181 PCT/USOO/04243 -102 After cooling at room temperature, 20 ptL of the master mix were added to each of the 21 solutions, in triplicate, and they were heated at 37 0 C for 15 minutes then placed on ice. 5 Five microliter samples of the solutions were placed in wells of a microtiter plate such that a 5 VL sample of each solution, in triplicate, was present within each plate and three such plates were prepared. The plates were placed into a Luminoskan@ 10 microtiter plate reading luminometer and this instrument was programmed to add 100 ptL of L/L reagent (Promega, F120B) to each well and immediately read the light produced by the reaction in the well. The individual readings for each solution within each 15 plate were averaged and these averages are given below. Relative Light Units Average Target Probe(s) Plate 1 Plate 2 Plate 3 of Plates none FV5 5.08 2.81 2.99 3.63 none FV6 2.85 2.72 3.59 3.05 none CV2 2.91 2.73 2.60 2.75 none FV6 and 2.56 2.75 2.68 2.66 CV2 9162+ none 2.67 2.59 2.50 2.59 9165 9163+ none 2.72 2.59 2.51 2.61 9166 WO 00/49181 PCT/USOO/04243 -103 FV(1+2) none 2.80 2.52 2.55 2.62 FV(3+4) none 2.75 2.41 2.51 2.56 9162+ none 2.57 2.53 2.34 2.48 9165+ FV (1+2 ) 9162+ none 2.54 2.46 2.40 2.47 9165+ FV(3+4) 9163+ none 2.40 2.39 2.45 2.41 9166+ FV (1+2 ) 9163+ none 2.48 2.35 2.42 2.42 9166+ FV(3+4) none none 2.53 2.34 2.22 2.36 9162+ FV5 25.61 28.23 24.08 25.97 9165+ FV (1+2 ) 9162+ FV6 and 4.75 4.53 4.32 4.53 9165+ CV2 FV (1+2 ) 9163+ FV5 25.36 27.72 28.98 27.35 9166+ FV (1+2 ) 9163+ FV6 and 44.69 41.14 45.29 43.71 9166+ CV2 FV (1+2 ) 9162+ FV5 3.91 3.93 4.16 4.00 916 5+ FV(3+4) WO 00/49181 PCT/USOO/04243 -104 9162+ FV6 and 32.23 30.57 36.55 33.12 9165+ CV2 FV(3+4) 9163+ FV5 3.54 3.64 3.52 3.57 9166+ FV(3+4) 9163+ FV6 and 58.61 59.14 71.77 63.17 9166+ CV2 FV(3+4) The light values for the reactions were adjusted from the averaged plate values above by subtracting the average No-DNA signal value and 5 target-alone averages and probe-alone values from the total light value measured for the various target and probe combinations. Reactions involving combinations of Target/Probe were further corrected by subtracting the appropriate adjusted probe-alone and target-alone 10 values to yield a net light value. The resulting values are shown in the table below. Targets FV5 Probes Mutant Probes WT CMV, WT Factor V 22.22 1.76 Mutant CMV, 23.68 41.00 WT Factor V WT CMV, 0.27 30.35 Mutant Factor V Mutant CMV, (-.12) 60.46 Mutant Factor V WO 00/49181 PCT/USOO/04243 -105 As in the previous example, a very distinctive signal pattern is seen with the various target combinations that were studied. This indicates that using multiple mutant probes in a 5 multiplex manner can reduce the number of reactions needed to determine if a mutant site is present within the sample. These data show for this assay system that when the signal from the mutant probe reactions approaches or is greater than that seen 10 with the corresponding wild type probe, the sample contains a target with a mutation in at least one of the sites. In addition, if the signal for the wild type (WT) probe is far lower than that for the multiplexed mutant probes, it is likely that at least 15 the target interrogated by the wild type probe is in the mutant form. FV1 5'CTAATCTGTAAGAGCAGATCCCTGGACAGGCGAGGAATACAGAGGGCAGCA 20 GACATCGAAGAGCT 3' SEQ ID NO:10 FV2 5'AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTCGCCTGTCCAGGGATCTG CTCTTACAGATTAGAGCT 3' SEQ ID NO:11 25 FV3 5'CTAATCTGTAAGAGCAGATCCCTGGACAGGCAAGGAATACAGAGGGCAGCA GACATCGAAGAGCT 3' SEQ ID NO:12 WO 00/49181 PCT/USOO/04243 -106 FV4 5'AGCTCTTCGATGTCTGCTGCCCTCTGTATTCCTTGCCTGTCCAGGGATCTG CTCTTACAGATTAGAGCT 3' SEQ ID NO:13 5 FV5 5' CTGCTGCCCTCTGTATTCCTCG 3' SEQ ID NO:14 FV6 5' CTGCTGCCCTCTGTATTCCTTG 3' SEQ ID NO:15 9162 5' CGTGTATGCCACTTTGATATTACACCCATGAACGTG CTCATCGACGTCAACCCGCACAACGAGCT 3' SEQ ID NO:16 10 9165 5' CGTTGTGCGGGTTCACGTCGATGAGCACGTTCATGG GTGTAATATCAAAGTGGCATACACGAGCT 3' SEQ ID NO:17 9163 5' CGTGTATGCCACTTTGATATTACACCCGTGAACGTG 15 CTCATCGACGTCAACCCGCACAACGAGCT 3' SEQ ID NO:18 9166 5'CGTTGTGCGGGTTCACGTCGATGAGCACGTTCACGG GTGTAATATCAAAGTGGCATACACGAGCT 3' SEQ ID NO:19 20 CV2 5' CACTTTGATATTACACCCGTG 3' SEQ ID NO:20 Example 3: Specific Detection of RNA: Comparison of Signals from RNA Species that Match Probe Sequences in 25 Reactions With and Without Added Extraneous Target RNA For the pyrophosphorylation reaction using DNA probes that match RNA sequences to be used to 30 detect specific target sequences, the system should give a very similar signal in the presence and absence of extraneous RNA. In this Example, the strength of the signal of probes designed to detect target globin mRNA in the presence of a large amount WO 00/49181 PCT/USOO/04243 -107 of yeast RNA is compared to the signal seen in the absence of added yeast RNA. Hybridization solutions containing various levels of yeast RNA, Probe 6 (SEQ ID NO:21) or Probe 8 (SEQ ID 5 NO:22) and target globin mRNA (Gibco BRL, 18103-028) were assembled by adding 5 piL of a 500 ng/ptL solution of either probe 6 or probe 8 to 5ptL of a 40 ng/iL solution of target globin mRNA and 10 ptL yeast RNA (Sigma Chemical Co. R3629) in 1X TE buffer (10 mM 10 Tris, 1 mM EDTA) to produce solutions containing total amounts of yeast RNA of 0, 2, 20, 200, 400, and 800 ng. The solutions were heated at 50 0 C for 15 minutes and then permitted to cool to room temperature for 15. 15 The following master reaction mixture was assembled: Nanopure water 346.5 piL MMLV-RT 5X Reaction Buffer (Promega 132 tL M195A) Sodium pyrophosphate (Promega M531) 16.5 tL NDPK (1 U/pL) 33 pL ADP (2 tM) 33 pL MMLV-RT (adjusted to 100 U/tL) 33 tL (Promega, M1701) The solution above was mixed and 18 ktL of 20 the mix were placed in 18 tubes. After cooling 15 minutes, 2 ptL of the various hybridization solutions WO 00/49181 PCT/USOO/04243 -108 containing probe 6 were added to the tubes and the tubes were placed in a 37'C heating block. After 15 minutes of incubation of the hybridization mixture with the reaction master mix, 5 20 pLL of the solution were added to 100 piL of L/L reagent (Promega, F202A) and the light output of the resulting reaction was measured using a Turner® TD 20/20 luminometer. After the probe 6 data were collected, an 10 identical set of reactions was performed using the hybridization solutions containing probe 8. The following data were obtained: Probe 6 Reactions Yeast RNA relative light units Average None 96 109 111 105.3 2 ng 98.4 85.0 118.5 100.7 20 ng 117.9 110.9 82.7 103.65 200 ng 56.4 110.1 93.2 86.6 400 ng 115.7 110.7 124.6 117 800 ng 127.6 128.7 143.1 133.1 Probe 8 Reactions Yeast RNA relative light units Average None 105.8 97.0 82.3 95.0 2 ng 84.5 84.6 93.7 87.6 20 ng 99.6 111.7 104.9 105.4 200 ng 83.6 75.9 95.6 85.1 400 ng 94.7 97.2 81.9 91.2 800 ng 50.7 89.0 82.1 73.9 WO 00/49181 PCT/USOO/04243 -109 These data indicate that addition of very large amounts of yeast RNA to the hybridization 5 reaction does not greatly lower the signal from hybridized probes for specific target RNA species. Probe 6 SEQ ID 5'AGACTTCTCCTCACTGGACAGATGCACC NO:21 AT3' Probe 8 SEQ ID 5'GGGTCCATGGGTAGACAACCAGCAGC3' NO:22 10 Example 4: Multiplex Analysis of Congenital Adrenal Hyperplasia (CAH) Gene The use of thermostable enzymes to interrogate the CAH gene permits the interrogation of 15 up to 6 multiple sites within one reaction. The method used in this Example is illustrative of routine studies carried out in screening laboratories where usual results show the presence of an expected gene (or the absence of a mutant gene) in almost all 20 of the samples, and only rarely shows the presence of a mutant gene. In the case illustrated here, a qualitative result is provided from which the exact mutation present can be determined in a subsequent assay. 25 Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive diseases resulting from a wide range of mutations in the steroid 21 hydroxylase (CYP21) gene that contains 10 exons.

WO 00/49181 PCT/USOO/04243 -110 There is a high level of nucleic acid homology (98% in exons, 96% in introns) between CYP21, the functional gene, and CYP21P, the nonfunctional pseudogene. The many types of mutations in this gene 5 that can lead to disease include complete gene deletions, large gene conversions, single point mutations, and a small 8bp deletion [See, White, et al., Hum. Mutat., 3:373-378, (1994)]. The majority of the CAH disease-causing 10 mutations are sequences present in the nonexpressed CYP21P pseudogene, and arise in the CYP21 gene through recombination between CYP21P and CYP21. Thus, one mutation detection strategy specifically detects the CYP21 gene, and not the CYP21P 15 pseudogene. The frequency of disease-carrying alleles in the population is about 1 in 50. Both wild type CAH PCR products, mutant synthetic targets, and a pseudogene PCR product amplified from the cloned CYP21P pseudogene were utilized as targets 20 in this assay. They are listed below. Primer pairs used in PCR amplification and the resulting products are as follows. Size PCR Segment 25 Primers Segment Amplified 10912 + 10909 1400 bp 5' end CYP21 11461 + 11480 918 bp 5' end CYP21 10910 + 11286 1492 bp 3' end CYP21 11535 + 11286 1496 bp 3' end CYP21 30 10912 + 10911 2680bp pseudogene (CYP21P) WO 00/49181 PCT/USOO/04243 -111 Synthetic targets and interrogation oligos utilized are listed below. PCR reactions were assembled to amplify regions of the CAH gene with 4 different probe sets, using 5 undigested human genomic DNA (Promega, G3041) as target (25 ng per reaction). For amplification of the pseudogene, human genomic DNA was predigested with the restriction enzyme Bcl I, which specifically cleaves the CYP21 gene upstream of the forward PCR 10 probe, thus permitting only amplification of CYP21P [Krone, Clinical Chem. 44(10) :2075-2082 (1998)]. The 2680 bp PCR product was amplified from 50 ng of digested DNA and subsequently cloned into the plasmid vector pGEM-T Easy (Promega, A1380) following 15 the manufacturer's protocol. A clone was selected and sequenced (USB Sequenase kit, US70770) to confirm it was indeed the pseudogene. The cloned CYP21P gene in the pGEM-T Easy vector was used in subsequent amplifications to obtain pure pseudogene PCR product 20 for mutation interrogation analysis (100 pg of plasmid per PCR reaction). All 50 tL amplification reactions contained the following reagents: genomic DNA (as described above), 1X reaction buffer (M1901), 1.0 - 1.5 mM 25 magnesium chloride (all with 1.0 mM except probe pair 10912+10911 for pseudogene, which contained 1.5 mM MgCl 2 ; Promega, A3511), 200 [tM each dNTP (C1141), 50 pmoles each probe, and 2.5 units Taq DNA Polymerase (M1665).

WO 00/49181 PCT/USOO/04243 -112 The following cycling profile was utilized for all amplifications: 5 minutes at 951C; 40 cycles of 30 seconds at 94 0 C, 1 minute at 55 0 C, 1 minute per kbp of product at 72 0 C; 8 minutes at 68oC; soak at 5 4 0 C. The products were analyzed on 1% agarose gels and compared to DNA molecular weight standards to confirm product sizes were correct. An aliquot of each PCR reaction (25 ptL) was then treated with 50 units T7 Gene6 Exonuclease (USB, E70025Y) for 15 10 minutes at 37 0 C, followed by purification using the Wizard' m PCR Prep DNA Purification System (Promega, A7170) with 3 x 1mL 80% isopropanol washes. The exonuclease-treated DNA was eluted in 100 tL of nuclease-free water. 15 Equal volumes of the CAH wild type (WT) 918 bp and 1496 bp PCR products were combined (to thus span the entire CAH gene) and interrogated either separately at each mutation site, or as a multiplexed group. 20 Each interrogation assay (20 tL total volume) contained 4 piL of purified PCR product or 5 ng of synthetic target, and 1 tg interrogation oligo probe (or water for the no-oligo background control). The reactions were incubated at 95 0 C for 3 minutes, 25 followed by 10 minutes at 37 0 C for Klenow exo- or 55 0 C for Tne polymerase. Twenty microliters of master mix were added (2 mM sodium pyrophosphate, 0.2 pM ADP, 2X polymerase buffer (M195A for Klenow or M1901 for Tne), 5 mM magnesium chloride for Tne only, 30 1-2 U Klenow exo- and 0.2 U yeast NDPK or 1 U Tne WO 00/49181 PCT/U S00/04243 -113 triple mutant polymerase and 0.1 U Pfu NDPK) and the reaction incubated 15 minutes at 371C (Klenow exo-) or 55 0 C (Tne). The entire reaction was then added to 100 tL of L/L reagent (Promega FF202A) and light 5 output read in a Turner® TD20/20 luminometer. The discrimination ratio was good both in the separate reactions for the combined PCR products, as well as the multiplexed reaction. In addition, the multiplexed reaction using the CAH wild type PCR 10 products and either 6 wild type interrogation oligo probes or 6 mutant interrogation oligo probes was combined with an equimolar amount of synthetic target (mutant synthetic target for each mutation site; 0.2 pmoles either PCR product or synthetic target), to 15 simulate a heterozygote sample. Tne/Pfu Tne/Pfu Tne/Pfu Probe Mutant Target NDPK, NDPK, NDPK for Synthetic DNA No WT Mutant Mutation Target Oligo Oligo Oligo Site Added CAH WT 918 bp + 172.7 553.0 180.2 1 1496 bp Same 172.7 535.7 184.0 2 Same 172.7 494.8 182.0 3 Same 172.7 486.7 148.7 4 Same 172.7 471.7 187.9 5 Same 172.7 317.5 179.7 6 Same 172.7 297.5 246.4 7 Same 523.7 1929.0 499.5 1,2,3,4, 5 and 6 WO 00/49181 PCT/USOO/04243 -114 Same 506.0 1882.0 2234.0 1 1 Same 525.4 1848.0 1505.0 2 2 Same 535.9 1735.0 2877.0 3 3 Same 547.5 1880.0 4879.0 4 4 Same 552.4 2000.0 3864.0 5 5 Same 482.9 1938.0 2189.0 6 6 Same 514.5 1791.0 4192.0 2 + 4 2 + 4 Same 537.6 1752.0 3427.0 5 + 6 5 + 6 Because of the large size of the CAH gene and the large number of different mutations that may be present, the use of the thermostable enzymes, and 5 thus the increased stringency of the detection procedure, was found to be highly advantageous with this complex target. Mutation sites that interrogated poorly using Klenow exo- and yeast NDPK at 370C, were more successfully interrogated when 10 using the Tne triple mutant polymerase and Pfu NDPK at elevated temperatures. In addition, use of the thermostable enzymes permitted the multiplexing of numerous wild type or mutant interrogation oligos in the same interrogation assay, to obtain the rapid 15 screening for mutations that may be present. PCR PRIMERS UTILIZED: 10909 5' CCAGAGCAGGGAGTAGTCTC 3' SEQ ID NO:66 CAH reverse primer; 5' most 3 linkages 20 phosphorothioate (CYP21 only) 10912 5' GCATATAGAGCATGGCTGTG 3' SEQ ID NO:67 CAH forward primer WO 00/49181 PCT/USOO/04243 -115 10910 5' CCTGTCCTTGGGAGACTAC 3' SEQ ID NO:68 CAH forward primer (CYP21 only) 10911 5' CCCAGTTCGTGGTCTAGC 3' SEQ ID NO:69 5 CAH reverse primer; 5' most 3 linkages phosphorothioate 11286 5' TCCTCACTCATCCCCAAC 3' SEQ ID NO:70 CAH reverse primer; 5' most 3 linkages 10 phosphorothioate 11461 5'GAAATACGGACGTCCCAAGGC SEQ ID NO:71 CAH forward primer 15 11480 5'CTTTCCAGAGCAGGGAGTAG SEQ ID NO:72 CAH reverse primer; 5' most 3 linkages phosphorothioate (CYP21 only) 11535 5'CCGGACCTGTCCTTGGGAGA SEQ ID NO:73 20 CAH forward primer (CYP21 only) SYNTHETIC TARGETS UTILIZED: 11293 5' AGAAGCCCGGGGCAAGAGGCAGGAGGTGGAGGCTCCGGAG 3' 25 SEQ ID NO:74 CAH Synthetic Target 1 for Interrogator oligo 1 (pseudogene/mutant - exon 1) Mutation site 1 30 11294 5' AGCTTGTCTGCAGGAGGAGCTGGGGGCTGGAGGGTGGGAA 3' SEQ ID NO:75 WO 00/49181 PCT/USOO/04243 -116 CAH Synthetic Target 2 for Interrogator oligo 2 (pseudogene/mutant - intron 2) Mutation site 2 5 11295 5' TCCGAAGGTGAGGTAACAGTTGATGCTGCAGGTGAGGAGA 3' SEQ ID NO:76 CAH Synthetic Target 3 for Interrogator oligo 3 (pseudogene/mutant - exon 4) Mutation site 3 10 11296 5' TCCACTGCAGCCATGTGCAAGTGCCCTTCCAGGAGCTGTC 3' SEQ ID NO:77 CAH Synthetic Target 4 for Interrogator oligo 4 (pseudogene/mutant - exon 7) 15 Mutation site 4 11297 5' TCGTGGTCTAGCTCCTCCTACAGTCGCTGCTGAATCTGGG 3' SEQ ID NO:78 CAH Synthetic Target 5 for Interrogator oligo 5 20 (pseudogene/mutant - exon 8) Mutation site 5 11298 5' GCTAAGGGCACAACGGGCCACAGGCGCAGCACCTCGGCGA 3' SEQ ID NO:79 25 CAH Synthetic Target 6 for Interrogator oligo 12 (pseudogene/mutant - exon 8) Mutation site 6 11484 5'CAGCTTGTCTGCAGGAGGAGTTGGGGGCTGGAGGGTGGGA 3' 30 SEQ ID NO:80 WO 00/49181 PCT/USOO/04243 -117 CAH Synthetic Target 7 for Interrogator oligo 7 (wild type - intron 2) Mutation site 2 5 11485 5'GGCTAAGGGCACAACGGGCCGCAGGCGCAGCACCTCGGCG 3' SEQ ID NO:81 CAH Synthetic Target 8 for Interrogator oligo 11 (wild type - exon 8) Mutation site 6 10 INTERROGATION OLIGOS PROBES UTILIZED: 11143 5' CGGAGCCTCCACCTCCCG SEQ ID NO:23 CAH interrogator oligo 6 (wild type) for mutation site 1 15 11085 5' CACCCTCCAGCCCCCAGC 3' SEQ ID NO:24 CAH interrogator oligo 2 (pseudogene/mutant) for mutation site 2 20 11084 5' CGGAGCCTCCACCTCCTG 3' SEQ ID NO:25 CAH interrogator oligo 1 (pseudogene/mutant) for mutation site 1 11086 5' CCTCACCTGCAGCATCAAC 3' SEQ ID NO:26 25 CAH interrogator oligo 3 (pseudogene/mutant) for mutation site 3 11144 5' CACCCTCCAGCCCCCAAC 3' SEQ ID NO:27 CAH interrogator oligo 7 (wild type) for mutation 30 site 2 WO 00/49181 PCT/USOO/04243 -118 11145 5' CCTCACCTGCAGCATCATC 3' SEQ ID NO:28 CAH interrogator oligo 8 (wild type) for mutation site 3 5 11087 5' CCTGGAAGGGCACTT 3' SEQ ID NO:29 CAH interrogator oligo 4 (pseudogene/mutant) for mutation site 4 11146 5' CCTGGAAGGGCACGT 3' SEQ ID NO:30 10 CAH interrogator oligo 9 (wild type) for mutation site 4 11088 5' GATTCAGCAGCGACTGTA 3' SEQ ID NO:31 CAH interrogator oligo 5 (pseudogene/mutant) for 15 mutation site 5 11147 5' GATTCAGCAGCGACTGCA 3' SEQ ID NO:32 CAH interrogator oligo 10 (wild type) for mutation site 5 20 11287 5' CGAGGTGCTGCGCCTGCG 3' SEQ ID NO:33 CAH interrogation oligo 11 (wild type) for mutation site 6 25 11288 5'CGAGGTGCTGCGCCTGTG 3' SEQ ID NO:34 CAH interrogation oligo 12 (pseudogene/mutant) for mutation site 6 11641 5'GGGATCACATCGTGGAGATG 3' SEQ ID NO:35 30 CAH interrogation oligo 23 (wild type) for mutation site 7 WO 00/49181 PCT/US0O/04243 -119 11642 5'GGGATCACAACGAGGAGAAG 3' SEQ ID NO:36 CAH interrogation oligo 24 (pseudogene/mutant) for mutation site 7 5 Example 5: HPLC Separation of dNTPs After Interrogation Assay, but Prior to Phosphate Transfer and Light Production 10 Large-volume pyrophosphorylation assays were performed on matched and mismatched probe/target hybrids. The released nucleotides were separated by high performance liquid chromatography (HPLC) and their fractions collected. NDPK terminal phosphate 15 transfer reactions were performed on these concentrated fractions and luciferase assays conducted to illustrate discrimination between the original matched and mismatched hybrid treated samples. 20 Target/probe hybrids were formed by combining 315 ng of the synthetic wild type CMV target oligonucleotide with either 10.5 ig wild type CMV probe for a matched hybrid, or 10.5 pig mutant CMV probe for a mismatched hybrid, and adding water to a 25 final volume of 200 ptL. The oligonucleotides were CV 12 (SEQ ID NO:37), CV 15 (SEQ ID NO:38), and CV 16 (SEQ ID NO:39). CV12 (SEQ ID NO:37) is a single stranded DNA segment of the genome of cytomegalovirus (CMV) in a form sensitive to the drug gancyclovir. 30 CV 15 (SEQ ID NO:38) is a probe that hybridizes with total complementarity to the non-resistant CMV. CV 16 (SEQ ID NO:39) is identical to CV 15 (SEQ ID WO 00/49181 PCT/USOO/04243 -120 NO:38) except that it contains a one base change from the CV15 sequence at the site of the SNP that confers drug resistance to the virus. These solutions were heated to 95 0 C for at least 5 minutes, then cooled at 5 room temperature for at least 10 minutes. The following master mix was prepared. 337.5 pL Nanopure water (Promega, AA399) 90.0 tL 1OX DNA Polymerase buffer (Promega, 10 M195A) 11.25 pL 40 mM NaPPi (Promega, C113) Master mix (210 pL) was added to each of the above hybrid solutions and 5.8 units of Klenow 15 exo- (Promega, M218A) were added to each. The solutions were then incubated at 37 0 C for 15 minutes and stored on ice. HPLC separation of the dNTPs was performed. HPLC separation of dATP, dCTP, dGTP and TTP 20 was performed on a 100 X 4.6 mm, 3 p Luna C18 column [Perrone and Brown, J. Chromatography, 317:301-310 (1984)] from Phenomenex. The column was eluted with a linear gradient of 97 percent buffer A (100 mM triethylammonium acetate, pH 7) to 92 percent buffer 25 A over a period of 12 minutes. The composition of buffer B is 80:20 acetonitrile:35 mM triethylammonium acetate. Detection was monitored by absorbance at 250, 260 and 280 nm. Under these conditions, dCTP was found to elute between 4 and 4.5 minutes, TTP and WO 00/49181 PCT/USOO/04243 -121 dGTP eluted as two peaks between 7 and 7.5 minutes, and dATP eluted from 9 to 9.5 minutes. The fractions containing the free dNTPs were collected and lyophilized. Fraction one 5 contained dCTP, fraction two contained dGTP and TTP, and fraction three contained dATP. Each fraction was reconstituted in 100 iL of nanopure water. Ten microliters of each fraction, or 10 tL of water as a control, were added to a 10 tL 10 mixture of water, 1oX DNA Polymerase Buffer, and ADP so that the final concentration was 1X DNA pol buffer and 0.1 FtM ADP. NDPK (0.005 units) was added to each tube in one set of the tubes and an equal amount of water was added to each tube in the other set of 15 tubes. Samples and controls were incubated at 37'C for 15 minutes, 10 LL added to 100 piL of L/L reagent and the light output was measured on a Turner® TD10/20 luminometer. The relative light units (rlu) results obtained are shown below: 20 Avg Sample Trial 1 Trial 2 Trial 3 rlu Matched hybrid with NDPK Fraction 1 206.6 200.6 205.9 204.4 25 Fraction 2 839.4 851.6 833.9 841.6 Fraction 3 1149.0 1150.0 1169.0 1156 Mismatched hybrid with NDPK Fraction 1 101.8 97.0 98.9 99.9 Fraction 2 386.1 387.3 382.2 385.2 30 Fraction 3 412.4 409.9 416.5 412.9 Match hybrid without NDPK Fraction 1 6.8 6.5 -- 6.6 Fraction 2 10.9 11.5 -- 11.2 WO 00/49181 PCT/USOO/04243 -122 Fraction 3 33.0 37.8 -- 35.4 Mismatched hybrid without NDPK Fraction 1 6.2 6.7 -- 6.4 Fraction 2 8.3 8.4 -- 8.4 5 Fraction 3 13.4 13.5 -- 13.4 No dNTP 7.9 7.5 -- 7.7 As is seen from the above data, the fraction one match:mismatch ratio is 2.1, fraction 2 10 match:mismatch ratio is 2.2 and fraction 3 match:mismatch ratio is 2.8. The data therefore demonstrate the utility of using HPLC separation of individual nucleotides followed by NDPK conversion to ATP, the preferred substrate of luciferase. Fraction 15 3 provides a slightly higher match:mismatch ratio owing to the presence of dATP in the nucleotide HPLC fraction. Nevertheless, HPLC separation of identifier nucleotides is useful in the interrogation assays of the present invention. 20 CV12 5' CCAACAGACGCTCCACGTTCTTTCTGACGTATTCGTGCAGCATGGTCTGCG AGCATTCGTGGTAGAAGCGAGCT 3' SEQ ID NO:37 25 CV15 5' CTACCACGAATGCTCGCAGAC 3' SEQ ID NO:38 CV16 5' CTACCACGAATGCTCGCAGAT 3' SEQ ID NO:39 WO 00/49181 PCT/USOO/04243 -123 Example 6: Multiplex Determination of Nucleotide Sequences Associated with Factor V Leiden and with a Prothrombin SNP in the Same Reaction 5 A assay is performed in this Example to determine if a human DNA sample contains the Leiden mutation of the Factor V gene, as well as a particular Prothrombin single nucleotide polymorphism (SNP). The assay for these two characteristics is 10 performed simultaneously in the same reaction. Probes PT5 (SEQ ID NO:40) and PT6 (SEQ ID NO:41) were used to PCR-amplify a region of human genomic DNA spanning about 500 base pairs encoding the prothrombin gene. Probes 10861 (SEQ ID NO:42) 15 and 9828 (SEQ ID NO:43) were used to PCR amplify a region of human genomic DNA spanning about 300 base pairs encoding the Factor V gene. Probes PT5 and 10861 have phosphorothioate linkages between the first five bases at the 5' end. 20 The Factor V and Prothrombin fragments were co amplified in one PCR reaction under the following conditions: 5 tL 1OX PCR buffer 25 5 ptL 25 mM MgCl 2 1 tL 10 mM dNTPs 1 plL probe PT5 (50 pmol) 1 pL probe PT6 (50 pmol) 1 ptL probe 10861 (50 pmol) 30 1 IlL probe 9828 (50 pmol) 1 plL Human genomic DNA (40 ng) WO 00/49181 PCT/USOO/04243 -124 36 pL water 1.25 U Taq The PCR cycling parameters were as follows: 5 940C, 2 minutes; (940C, 30 seconds; 60'C, 1 minutes; 70'C, 1 minutes) x 40; 70'C, 5 minutes. Fifty units of T7 gene 6 Exonuclease (USB Amersham) were added to 25 ptL of the PCR reaction and the solution was incubated for 30 minutes at 37 0 C. Magnetic silica 10 (Promega, A1330) was used to remove free nucleotides from the solution and the remaining DNA was eluted with 100 tL of water. The Prothrombin interrogation probes used are 11265 (SEQ ID NO:44) that matches mutant 15 prothrombin sequence and 11266 (SEQ ID NO:45) that matches wild type prothrombin sequence. Each of those probes has a destabilizing mutation eight bases from the 3' end. The Factor V interrogation probes used are 9919 (SEQ ID NO:46) that matches wild types 20 Factor V sequence and 11432 (SEQ ID NO:47) that matches Factor V Leiden mutation sequence. Four microliters of the eluted DNA were interrogated with each interrogation probe independently and also with the Factor V and 25 Prothrombin mutant probes conjointly in one reaction. The interrogation reactions were assembled as follows. 4 ptL DNA (PCR product, Exo6 treated and 30 purified) WO 00/49181 PCT/USOO/04243 -125 150 pmol each interrogation oligo water added to a final volume of 20 piL The reactions were incubated at 95 0 C for 3 5 minutes and then at 37 0 C for 10 minutes. Twenty microliters of the standard master mix was then added and the reaction incubated at 37'C for 15 minutes. One hundred microliters of the L/L reagent were then added and the light output measured in a Turner® 10 TD20/20 luminometer. The master mix contains the following. 71 pL water 20 tL 1OX DNA pol buffer 15 5 pIL 40 mM NaPPi 2 tL 10 .tM ADP 1 piL 1 unit/pL NDPK 1 10 unit/tL Klenow exo 20 The light output was as follows. Interrogation Genomic Genomic oligo DNA 1 DNA 2 25 9919 (FV wt) 431 424 11432 (FV mut) 45 57 11266 (Pt wt) 902 878 11265 (Pt mut) 145 161 11432 + 11265 77 98 30 no oligo 44 57 WO 00/49181 PCT/USOO/04243 -126 These data indicate that the both genomic DNAs are from individuals wild type for Factor V and for wild type Prothrombin. 5 An additional 96 clinical genomic DNA samples were interrogated as described above. All the data fit into the following equation for calling the genotype. rlu both wild type probes >0.75 10 rlu both wild type + rlu both mutant probes This equation is the analytical output from the interrogation including both wild type probes divided by the analytical output from both wild type probes 15 added to the analytical output from both mutant probes. If that value is greater than 0.75 then the sample is homozygous wild type at both loci. If that value is less than 0.75 then there is good likelihood that at least one allele at least one of the loci is 20 mutant and the sample should be further analyzed for the genotype at each locus separately. PT5 5' ATAGCACTGGGAGCATTGAGGC 3' SEQ ID NO:40 25 PT6 5' GCACAGACGGCTGTTCTCTT 3' SEQ ID NO:41 10861 5' TGCCCAGTGCTTAACAAGACCA 3' SEQ ID NO:42 9828 5' TGTTATCACACTGGTGCTAA 3' SEQ ID NO:43 30 11265 5' GTGATTCTCAGCA 3' SEQ ID NO:44 WO 00/49181 PCT/USOO/04243 -127 11266 5' GTGATTCTCAGCG 3' SEQ ID NO:45 9919 5' GACAAAATACCTGTATTCCTCG 3' SEQ ID NO:46 5 11432 5' GACAAAATACCTGTATTCCTTG 3' SEQ ID NO:47 Example 7: Multiplex Interrogation for Factor V Leiden and Prothrombin Mutation: 10 Mass Spectroscopy Analysis This Example demonstrates that nucleotides released from the 3'-terminus of a hybridized probe in a multiplex reaction by a process of the invention can be detected by mass spectroscopy and thereby 15 determine whether a mutant allele exists at one of the loci being studied. Probes PT5 (SEQ ID NO:40) and PT6 (SEQ ID NO:41) are used to PCR-amplify a region of human genomic DNA spanning about 500 base pairs encoding 20 the prothrombin gene. Probes 10861 (SEQ ID NO:42) and 9828 (SEQ ID NO:43) are used to PCR amplify a region of human genomic DNA spanning about 300 base pairs encoding the Factor V gene. These probes and the PCR reaction conditions are detailed in Example 25 6. Probes PT5 and 10861 have phosphorothioate linkages between the first five bases at the 5' end. The PCR product is treated with T7 gene 6 Exonuclease (USB Amersham) and separated from free nucleotides as described in Example 6. 30 The prothrombin interrogation probe, 11265 (SEQ ID NO:44), is totally complementary to a segment of the mutant prothrombin sequence. The Factor V WO 00/49181 PCT/USOO/04243 -128 interrogation probe, 11432 (SEQ ID NO:47), is totally complementary to a segment of the mutant Factor V Leiden mutation sequence. Each of these probes has a destabilizing mutation eight bases from the 3'-end. 5 The PCR products are synthesized, Exo 6 treated, and purified as described in Example 6. The interrogation reactions are assembled with 40 ptL of each PCR product and 1.5 nmol of each interrogation probe. Water is added to a final volume of 100 [tL. 10 These reactions are assembled in duplicate so that one can be assayed with Klenow exo- polymerase and yeast NDPK at 37 0 C, while the other is assayed with Tne triple mutant polymerase and Pfu NDPK at 70 0 C. These assembled reactions are incubated at 15 95 0 C for 3 minutes and then at 37 0 C for 10 minutes. The assembled reactions may be lyophilized to decrease the volume. The two different master mixes are assembled. Both master mixes have 2 mM sodium pyrophosphate and 0.2 pM ADP. One with 1-2 U Klenow 20 exo- and 0.2 U yeast NDPK, and 2X polymerase buffer (M195A); the other with 1 U Tne triple mutant polymerase and 0.1 U Pfu NDPK, 2X polymerase buffer (M1901), and 5 mM magnesium chloride. An equal volume of each master mix is separately added to the 25 reaction solutions described above. Then the solution containing Klenow exo- as the polymerase is incubated at 37 0 C for 15 minutes, while the solution containing Tne triple mutant polymerase is incubated at between 55 0 C and 70 0

C.

WO 00/49181 PCT/USOO/04243 -129 The presence or absence of released nucleotides, converted to ATP, is analyzed for by silicon desorption ionization mass spectroscopy (Wei, J. et al. Nature. 399:243-246, 1999). This method is 5 sensitive to femtomole and attomole levels of analyte. The samples are prepared as described in that paper. Essentially, analytes are dissolved in a deionized water/methanol mixture (1:1) at concentrations typically ranging from 0.001 to 10.0 10 pIM. Aliquots (at least 0.5 to 1.0 pL, corresponding to at least 0.5 femtomol to 100 picomol analyte) of solution are deposited onto the porous surfaces and allowed to dry before mass spectrometry analysis. These experiments are performed on a Voyager DE-STR, 15 time-of-flight mass spectrometer (PerSeptive Biosystems) using a pulsed nitrogen laser (Laser Science) operated at 337 nm. Once formed, ions are accelerated into the time-of-flight mass analyzer with a voltage of 20 kV. Other liquid 20 chromatography-mass spectrometry (LC-MS) instrumentation may also be used for analysis (Niessen W. J. Chromatogra A 794: (407-435, 1998). An observance of released nucleotide from either of the reactions containing the two mutant 25 probe, at levels greater than background, indicates the presence of a at least one mutant prothrombin or Factor V Leiden allele in the genomic DNA sample assayed. 30 10861 5' TGCCCAGTGCTTAACAAGACCA 3' SEQ ID NO:42 9828 5' TGTTATCACACTGGTGCTAA 3' SEQ ID NO:43 WO 00/49181 PCT/USOO/04243 -130 PT5 5' ATAGCACTGGGAGCATTGAGGC 3' SEQ ID NO:40 PT6 5' GCACAGACGGCTGTTCTCTT 3' SEQ ID NO:41 11265 5' GTGATTCTCAGCA 3' SEQ ID NO:44 11432 5' GACAAAATACCTGTATTCCTTG 3' SEQ ID NO:47 5 Example 8: Multiplex Interrogation using Fluorescent Labels This example demonstrates that nucleotides released from the 3'-terminus of multiple probes, 10 each hybridized to a target nucleic acid of interest, by a process of the invention can be detected by mass spectrometry or by fluorimetric HPLC and thereby provide evidence of the presence or absence of the target nucleic acid in a nucleic acid sample or of a 15 specific base at an interrogation position of the target. Each interrogation probe is designed to have a different fluorescent label attached to the 3'-terminal nucleotide in a manner such that the 20 label does not interfere with the ability of the depolymerizing enzyme to remove the nucleotide from the probe. Such fluorescent tags, such as fluorescein or rhodamine, can be incorporated into the probe during synthesis with a fluorescent 25 molecule attached to the phosphoramadite nucleotide with a linker of at least 6 carbons (Glen Research). Probes PT5 (SEQ ID NO:40) and PT6 (SEQ ID NO:41) are used to PCR-amplify a region of human genomic DNA spanning about 500 base pairs encoding 30 the prothrombin gene. Probes 10861 (SEQ ID NO:42) and 9828 (SEQ ID NO:43) are used to PCR amplify a region WO 00/49181 PCT/USOO/04243 -131 of human genomic DNA spannning about 300 base pairs encoding the Factor V gene. These probes and the PCR reaction conditions are detailed in Example 6. The PCR products are treated with T7 gene 6 Exonuclease 5 (USB Amersham) and separated from free nucleotides as described in Example 6. The prothrombin interrogation probes are 11265 (SEQ ID NO:44), that is totally complementary to a segment of the mutant prothrombin sequence, and 10 11266 (SEQ ID NO:45), that is totally complementary to a segment of the wild-type prothrombin sequence. Each of these probes has a destabilizing mutation eight bases from the 3 1 -end. Also, each of these probes is synthesized with a fluorescent nucleotide 15 analog at the 3'-terminal nucleotide position. The prothrombin probes are tagged with fluorescein; the factor V probes are tagged with rhodamine. The purified PCR products are interrogated in separate reactions with either both wild-type 20 probes or both mutant probes. Interrogation reactions are assembled as follows: 40 tL each of the two PCR products 1.5 nmol each of the wild type or each of the mutant labeled interrogation oligos 25 water is added to a final volume of 100 piL. The reactions are incubated at 95 0 C for 3 minutes and then at 37 0 C for 15 minutes. The reactions are then lyophilized to a final volume of 20 tL.

WO 00/49181 PCT/USOO/04243 -132 Twenty microliters of master mix are then added. The composition of the master mix containing Klenow exo- is described in Example 4 with the exception that there is no ADP and no NDPK. The 5 reaction then proceeds at 37 0 C for 15 minutes. The solutions are then split in half and analyzed using two different methods. In one method, the presence or absence of released nucleotides in the solutions is analyzed by silicon desorption 10 ionization mass spectroscopy (Wei, J. et al. Nature. 399:243-246, 1999). This method is sensitive to femtomole and attomole levels of analyte. The samples are prepared for spectrometry as described in that paper. Essentially, analytes are dissolved in a 15 deionized water/methanol mixture (1:1) at concentrations typically ranging from 0.001 to 10.0 [tM. Aliquots (at least 0.5 to 1.0 ptL, corresponding to at least 0.5 femtomol to 100 picomol analyte) of solution are deposited onto the porous surfaces and 20 permitted to dry before mass spectrometry analysis. These studies are performed on a Voyager DE-STR, time-of-flight mass spectrometer (PerSeptive Biosystems) using a pulsed nitrogen laser (Laser Science) operated at 337 nm. Once formed, ions are 25 accelerated into the time-of-flight mass analyser with a voltage of 20 kV. Other liquid chromatography-mass spectrometry (LC-MS) instrumentation may be used for analysis (Niessen W. J. Chromatogra A 794:407-435, 1998) 30 In a second method, the presence or absence of released nucleotides in the solutions is analyzed WO 00/49181 PCT/US00/04243 -133 by HPLC using a fluorescence detector as described in Jain, et al. Biochem Biophys Res Commun 200:1239 1244, 1994 or Levitt, B. et al. Anal Biochem 137:93 100, 1984. 5 An observance of released nucleotide from the reactions containing the mutant probes, at levels greater than background (control reactions that contain no enzyme), is indicative of the presence of at.least one mutant prothrombin or Factor V Leiden 10 allele in the genomic DNA sample assayed. An observance of released nucleotide from the reaction containing the wild-type probes, at levels greater than background, is indicative of the presence of at least one wild-type prothrombin or Factor V allele in 15 the genomic DNA sample assayed. PT5 5' ATAGCACTGGGAGCATTGAGGC 3' SEQ ID NO:40 PT6 5' GCACAGACGGCTGTTCTCTT 3' SEQ ID NO:41 10861 5' TGCCCAGTGCTTAACAAGACCA 3' SEQ ID NO:42 20 9828 5' TGTTATCACACTGGTGCTAA 3' SEQ ID NO:43 Example 9: Speciation-Detection of Mitochondrial DNA Specific to Various Animals 25 In this example, a segment of mitochondrial DNA comprising a segment of the cytochrome B gene was amplified from a variety of animal species using PCR primers 11590 (SEQ ID NO:48) and 11589 (SEQ ID NO:49) (PNAS 86:6196-6200). These PCR primers were diluted 30 in 10mM Tris, pH 7.5, to a final concentration of 0.22 p.g/ tL. The genomic DNAs used were bovine WO 00/49181 PCT/USOO/04243 -134 (Clontech, 6850-1), chicken (Clontech, 6852-1), dog (Clontech, 6950-1) and human (Promega, G1521). The PCR reactions were assembled to include 5 tL 1oX buffer with 15 mM MgCl 2 (Promega, M188J), 1 5 tL dNTPs 10 mM (Promega, C144G), 2 tL primer 11590, 2 pLL primer 11589, 0.5 tL Taq polymerase 5U/tL (Promega, M186E), and 38.5 ptL water. To each tube was then added 1 tL (100 ng) of genomic DNA. The PCR cycling parameters were (15 seconds, 94'C; 15 seconds, 10 55 0 C; 30 seconds, 72'C) x 30. The size of PCR products was confirmed by running an aliquot on an agarose gel and visualizing with ethidium bromide (EtBr) staining. The PCR products were then separated from free nucleotides (Promega, A7170) and an aliquot 15 run on an agarose gel. All samples produced a PCR product of the same size. Each PCR DNA was then used in an assay to determine if it could be specifically identified with a species-specific probe. One microliter of 20 interrogation probe (1pg/p.L) and 17 piL water were combined with 2 tL of the appropriate PCR product and heated at 91 0 C for 3 minutes, then cooled at room temperature for 15 minutes. Twenty microliters of master mix (described below) were added to each tube 25 and each was further incubated at 370C for 15 minutes. Four microliters of the solutions were then added to 100 pIL L/L reagent (Promega F120B), and the relative light output (rlu) measured on a Turner® TD20/20 luminometer. The rlu average values from two WO 00/49181 PCT/USOO/04243 -135 reactions, minus the DNA background values, along with the standard deviation values are listed below. Master mix: 5 312 [pL 1OX DNA pol buffer (Promega M195A) 39 p.L NaPPi 40 mM (Promega E350B) 39 pL Klenow exo minus (Promega M128B) 15.6 [pL NDPK 1 U/[pL 31.2 [pL ADP 10 JIM (Sigma) 10 1123 [pL water (Promega AA399) Averages from 2 reactions. Net light units Standard Deviations are calculated by subtracting the DNA background No Human Chicken Cow Dog No Human Chicken Cow Dog Probe DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA comzoo -0.096 44.5 14.25 119.7 124.6 0.654 7.000 23.33 3.465 8.63 huzool 1.771 38 -40 -51.85 -63.55 0.137 38.96 31.74 6.505 12.52 huzoo2 -0.889 101.6 -23.35 -0.05 -48.75 0.761 3.959 0.141 8.768 2.19 chzool 43.07 -30.4 34.05 -2.75 -31.15 7.078 1.909 6.364 2.687 8.70 chzoo2 -0.361 57.6 50.7 33.05 -3.25 0.075 43.77 29.34 12.59 21.43 cozoo2 1.925 90.95 125.1 202.6 132.5 0.208 20.08 13.22 8.627 19.30 dozoo2 0.966 71.8 158.7 0.180 9.546 1.98 15 The data demonstrate that the primers detect the mitochondrial PCR product. Both of the human-specific probes (11576 (SEQ ID NO:50) and 11583 (SEQ ID NO:51) were shown to be specific for human mitochondrial DNA. The common probe, 11582 (SEQ ID 20 NO:52), detected all of the species, but was less WO 00/49181 PCT/USOO/04243 -136 efficient with chicken DNA. The chicken-specific probe, 11577 (SEQ ID NO:53), was specific for chicken mitochondrial DNA, but the other chicken-specific probe, 11584 (SEQ ID NO:54), detected all the species 5 except dog. The cow-specific probe, 11588 (SEQ ID NO:55), gave the best detection signal for cow DNA, but also detected the other species. The dog specific probe, 11586 (SEQ ID NO:56), was assayed only with dog and cow DNA, but detected the dog DNA 10 better than cow DNA. A cleaner PCR product provides DNA with less background. 11590 zooamp2 5' AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA 3' 15 SEQ ID NO:48 11589 zooampl 5' AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA 3' SEQ ID NO:49 20 11576 huzool 5' CCAGACGCCTCA 3' SEQ ID NO:50 11583 huzoo2 5' ACCTTCACGCCA 3' SEQ ID NO:51 25 11582 comzoo 5' TGCCGAGACGT 3' SEQ ID NO:52 11577 chzool 5' GCAGACACATCC 3' SEQ ID NO:53 11584 chzoo2 5' GGAATCTCCACG 3' SEQ ID NO:54 30 11588 cozoo2 5' ACATACACGCAA 3' SEQ ID NO:55 WO 00/49181 PCT/USOO/04243 -137 11586 dozoo2 5' ATATGCACGCAA 3' SEQ ID NO:56 Example 10: Self-annealing Interrogation Probe 5 This Example illustrates use of a different type of oligonucleotide probe that is used to form a hairpin structure in the interrogation technology of this invention. This study demonstrates a method for 10 eliminating the need for adding a probe specific to the interrogation site to the interrogation reaction. Here, the oligonucleotide probe anneals to the target strand downstream of (3' to) the interrogation position in the target strand. The 15 oligonucleotide has at its 5' end an unannealed region of nucleotides followed by about 5 to about 20 nucleotides that are identical to the interrogation region on the target strand. The annealed 3' end of the oligonucleotide is then extended through the 20 interrogation position of the target strand creating what is referred to as extended probe. The hybrid is denatured and a hairpin structure formed between the extended probe strand and the 5' end of the oligonucleotide probe. This region is then assayed in 25 a standard interrogation reaction to determine if a mismatch is present or not. Four probes were designed to represent different types of hairpin formations that an extended probe strands may assume. These probes are 30 10207 (SEQ ID NO:57), 10208 (SEQ ID NO:58), 10209 (SEQ ID NO:59), and 10212 (SEQ ID NO:60).

WO 00/49181 PCT/USOO/04243 -138 These probes are predicted to form the following self-hybridized secondary structures when allowed to self-anneal: 5 10207 5' A-T-G-A-A-C-G-T-A-C-G-T-C-GjG 3' T-A-C-T-T-G-C-A C-G-A-G-T-A 10208 5'G 10 T-G-A-A-C-G-T-A-C-G-T-C- G-G A-C-T-T-G-C-A 3'T C-G-A-G-T-A 15 10209 5' A-T A-A-C-G-T-A-C-G-T-C-G-G 3' T-A T-T-G-C-A C C-G-A-G-T-A 20 10212 5' A-T-A-A-A-C-G-T-A-C-G-T-C-G-G 3' G-C-A\ C-G-A-G-T-A 25 A 5 tL (5 ptg) aliquot of each of the four probes was diluted to 100 tL with nanopure water. They were then sequentially diluted 1:10 to a final dilution factor of 1:100,000. Twenty microliters of the diluted probes were heated, in 30 separate tubes, at 95 0 C for 3 minutes and cooled to room temperature for 10 minutes to permit self- WO 00/49181 PCT/USO0/04243 -139 annealing. The following master mix was assembled and mixed. Component Amount 1oX DNA Pol Buffer 200 tL (Promega, M195A) Klenow exo- (1U/pL) 12.5 pL (Promega M218B) 40 mM Sodium Pyrophosphate 25 ptL (Promega C350B) NDPK (1U/ptL) 10 pL 10uM ADP (Sigma A5285) 20 p.L Water 732.5 tL 5 Twenty microliters of the above Master Mix, were then added to each tube and the tubes were incubated at 37*C for 15 minutes. Ten microliters of the solutions were added to 100 pL of L/L reagent (Promega, F202A) and relative light units measured 10 immediately with a Turner® TD20/20 luminometer. The no-probe control resulted in 57.24 relative light units and the remaining probe results are reported below in relative light units (rlu). 15 Log Probe dilution 10207 10208 10209 10212 -5 44.89 56.22 57.57 57.80 -4 85.21 64.56 58.26 63.15 -3 297.7 70.53 79.12 82.65 20 -2 970.5 108.4 80.06 106.7 WO 00/49181 PCT/USOO/04243 -140 Probe 10207 worked as an efficient target for interrogation as expected, with probe 10208 providing the anticipated negative results. Probe 10212 has only a three base match so it may be un 5 extended, thus resulting in the low values. Probe 10209 likely has the 3' terminal nucleotide unannealed when the hairpin forms due to the mismatch at the third nucleotide in from the 3' end. Such an unannealed 3' terminal nucleotide would account for 10 the low rlu values. 10207 5' ATGAACGTACGTCGGATGAGCACGTTCAT 3' SEQ ID NO:57 15 10208 5' GTGAACGTACGTCGGATGAGCACGTTCAT 3' SEQ ID NO:58 10209 5' ATAAACGTACGTCGGATGAGCACGTTCAT 3' SEQ ID NO:59 20 10212 5' ATAAACGTACGTCGGATGAGCACG 3' SEQ ID NO:60 Example 11: Interrogation with a 25 Self-Annealing Primer II This example and Fig. 2 illustrate use of a different type of oligonucleotide probe, a "REAPER'" probe in a process of this invention. This example 30 demonstrates a method for eliminating the need for adding a probe specific to the interrogation site to the interrogation reaction.

WO 00/49181 PCT/USOO/04243 -141 Here, the oligonucleotide first probe (SEQ ID NO:62), at its 3'-end, anneals to the target strand (SEQ ID NO:61) at a position downstream of (3' to) the interrogation position in the target strand 5 (Fig. 2A). The probe has at its 5'-end an unannealed region of nucleotides including about 5 to about 20 nucleotides that are identical to a region on the target strand including the interrogation position. This region of identity is present in the same 10 orientation on both the target and the probe strands. The annealed 3'-end of the probe is then extended through the interrogation position of the target strand forming what is referred to as a first extended probe and an extended first hybrid as is 15 illustrated in Fig.2B (SEQ ID NO:63). The extended first hybrid is denatured and a second probe (SEQ ID NO:64) is annealed to the first extended probe to form a second hybrid. This second probe is complementary to the first extended probe strand at a 20 region downstream of the interrogation position on the first extended probe strand (Fig. 2C). The second probe is then extended and a second extended hybrid is formed as illustrated in Fig. 2D. The second extended hybrid is comprised of 25 the first extended probe and second extended probe (SEQ ID NO:65). The strands of the second extended hybrid are denatured and permitted to renature to form a hairpin structure. Upon hairpin formation, the first 30 extended probe forms a hairpin structure that has a 3'-overhang, whereas the second extended probe forms WO 00/49181 PCT/USOO/04243 -142 a hairpin structure that contains a 5'-overhang that provides a substrate for depolymerization. The second extended probe strand is then depolymerized and the analytical output obtained as described 5 elsewhere herein. The analytical output determines the presence or absence of the original target strand or of a particular base in the original target strand as is also discussed elsewhere herein. SEQ ID NO:61 oligonucleotide is diluted to 10 1 mg/mL in water. This solution is labeled 286. SEQ ID NO:62 oligonucleotide is diluted to 1 mg/mL in water and this solution is labeled 287. One microliter of each solution 286 and 287 is combined with 18 tL water. The solution is heated to 95 0 C for 15 5 minutes then is cooled at room temperature for 10 minutes to permit oligonucleotides of SEQ ID NOs:61 and 62 to anneal. To this solution are added dNTP mixture to a final concentration of 0.25 mM for each dNTP, 1oX 20 Klenow buffer to a final concentration of 1X, and 5 U of Klenow enzyme. The tube with these components is incubated at 37'C for 30 minutes. The extended first hybrid DNA so formed (containing SEQ ID NO: 63) is purified (Qiagen, Mermaid system) and eluted into 50 25 ptl of water. To this solution of the purified extended first hybrid is added 1 pl SEQ ID NO: 64 oligonucleotide (1 mg/mL) as second probe. The solution is then heated to 95 0 C for 5 minutes and is 30 cooled at room temperature to permit 289 and 288 to WO 00/49181 PCT/USOO/04243 -143 anneal as illustrated in Fig. 2C to form the second hybrid. To this solution are added a dNTP mixture to a final concentration of 0.25 mM for each dNTP, 1OX Klenow buffer to a final concentration of 1X, and 5 U 5 of Klenow enzyme. The tube with these components is incubated at 37'C for 30 minutes to form a second extended hybrid that contains a second extended probe (oligonucleotide SEQ ID NO: 65). The SEQ ID NO: 65/63 second extended hybrid 10 DNA (Fig. 2D) formed is purified (Qiagen, Mermaid system) to separate the extended hybrid from the unreacted dNTPs and eluted into 50 ptl water. (Alternatively, the original 287 oligo is biotinylated at it's 5'-end and this biotin is then 15 also present in strand of SEQ ID NO: 63. This biotinylated strand 288 is then denatured from strand 290 and removed from the solution with streptavidin coated paramagnetic particles according to the manufacturer's instructions (Promega, Z5481) and the 20 290 hairpin structure is allowed to form as below). This hybrid solution is then heated to 95*C for 5 minutes diluted to 100 pl with water and is cooled on ice for 10 minutes to permit hairpin structure formation. 25 A master mix is assembled and mixed according to the following table. Component Amount 1OX DNA Pol Buffer 200 pL (Promega, M195A) WO 00/49181 PCT/USOO/04243 -144 Component Amount Klenow exo- (1 U/pL) 12.5 PtL (Promega M218B) 40 mM Sodium Pyrophosphate 25 LL (Promega C350B) NDPK (1 U/tL) 10 tL 10uM ADP (Sigma A5285) 20 [L Water 732.5 ptL Twenty microliters of this master mix are added to 20 tL of the above hairpin-containing solutions after cooling, and the resulting mixtures 5 are heated at 37 0 C for 15 minutes. After this incubation, duplicate 4 piL samples of the solution are removed, added to 100 tL of L/L Reagent (Promega, F202A) and the light produced by the reaction is measured immediately using a Turner® TD20/20 10 luminometer. A positive analytical output at levels over background (no enzyme) indicates that a matched base was present at the 3'-terminus of the hairpin structure and this further indicates the presence of the target strand, and for this particular example, 15 it also indicates the presence of a G base at the interrogation position of the target. 5' CCCGGAGAGACCTCCTTAAGGGGCCATATTATTTCGTCGATTCCAGTGTT GGCCAAACGGAT 3' SEQ ID NO: 61 20 5' GGGGCCATATTATTTCGCCGTTTGGCCAACACTGGAATCGA 3' SEQ ID NO: 62 WO 00/49181 PCT/USOO/04243 -145 5' GGGGCCATATTATTTCGCCGTTTGGCCAACACTGGAATCGACGAAATAAT ATGGCCCCTTAAGGAGGTCTCTCCGGG 3' SEQ ID NO: 63 5 5' CCCGGAGAGACCTCCT 3' SEQ ID NO: 64 5' CCCGGAGAGACCTCCTTAAGGGGCCATATTATTTCGTCGATTCCAGTGTT GGCCAAACGGCGAAATAATATGGCCCC 3' SEQ ID NO: 65 10 From the foregoing, it will be observed that numerous modifications and variations can be effected without departing from the true spirit and scope of the present invention. It is to be understood that no limitation with respect to the 15 specific examples presented is intended or should be inferred. The disclosure is intended to cover by the appended claims modifications as fall within the scope of the claims.

Claims

1. A method for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample that 5 comprises the steps of: (A) providing a treated sample that may contain said plurality of predetermined nucleic acid target sequences hybridized with their respective nucleic acid probes, said probes each including an 10 identifier nucleotide in the 3'-terminal region; (B) admixing the treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3' terminus of a hybridized nucleic acid probe to form a 15 treated reaction mixture; (C) maintaining the treated reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom; and 20 (D) analyzing for the presence of released identifier nucleotides to obtain an analytical output, the analytical output indicating the presence or absence of said nucleic acid target sequences. 25

2. The method according to claim 1 wherein said analytical output is obtained by luminescence spectroscopy.

3. The method according to claim 1 30 wherein said analytical output is obtained by fluorescence spectroscopy. WO 00/49181 PCT/USOO/04243 -147

4. The method according to claim 1 wherein said analytical output is obtained by mass spectrometry. 5

5. The method according to claim 1 wherein said analytical output is obtained by absorbance spectroscopy. 10

6. The method according to claim 1 wherein said predetermined nucleic acid target sequences are associated with blood coagulation.

7. The method according to claim 6 15 wherein said nucleic acid probes comprise a plurality of the following sequences: 5' CTGCTGCCCTCTGTATTCCTCG 3' SEQ ID NO:14; 5' CTGCTGCCCTCTGTATTCCTTG 3' SEQ ID NO:15; 5' GTGACTCTCAGCG 3' SEQ ID NO:87; 20 5' GTGACTCTCAGCA 3' SEQ ID NO:88; 5' GTGATTCTCAGCG 3' SEQ ID NO:89; 5' GTGATTCTCAGCA 3' SEQ ID NO:90; 5' GACAAAATACCTGTATTCCTCG 3' SEQ ID NO:91; 5' GACAAAATACCTGTATTCCTTG 3' SEQ ID NO:92; 25 5' GGAGCATTGAGGCTCG 3' SEQ ID NO:93; 5' GGAGCATTGAGGCTTG 3' SEQ ID NO:94; 5' GACAAAATACCTGTATTCCTTG 3' SEQ ID NO:47; 5' GTGATTCTCAGCA 3' SEQ ID NO:44; 5' GTGATTCTCAGCG 3' SEQ ID NO:45; and 30 5' GACAAAATACCTGTATTCCTCG 3' SEQ ID NO:46. WO 00/49181 PCT/USOO/04243 -148

8. The method according to claim 1 wherein said predetermined nucleic acid target sequences are useful for speciation. 5

9. The method according to claim 8 wherein said nucleic acid probes comprise a plurality of the following sequences: 5' CCAGACGCCTCA 3' SEQ ID NO:50; 5' ACCTTCACGCCA 3' SEQ ID NO:51; 10 5' TGCCGAGACGT 3' SEQ ID NO:52; 5' GCAGACACATCC 3' SEQ ID NO:53; 5' GGAATCTCCACG 3' SEQ ID NO:54; 5' ACATACACGCAA 3' SEQ ID NO:55; and 5' ATATGCACGCAA 3' SEQ ID NO:56. 15

10. The method according to claim 1 wherein said predetermined nucleic acid target sequences are associated with congenital adrenal hyperplasia 20

11. The method according to claim 10 wherein said nucleic acid probes comprise a plurality of the following sequences: 5' CGGAGCCTCCACCTCCCG SEQ ID NO:23; 25 5' CACCCTCCAGCCCCCAGC 3' SEQ ID NO:24; 5' CGGAGCCTCCACCTCCTG 3' SEQ ID NO:25; 5' CCTCACCTGCAGCATCAAC 3' SEQ ID NO:26; 5' CACCCTCCAGCCCCCAAC 3' SEQ ID NO:27; 5' CCTCACCTGCAGCATCATC 3' SEQ ID NO:28; 30 5' CCTGGAAGGGCACTT 3' SEQ ID NO:29; 5' CCTGGAAGGGCACGT 3' SEQ ID NO:30; WO 00/49181 PCT/USOO/04243 -149 5' GATTCAGCAGCGACTGTA 3' SEQ ID NO:31; 5' GATTCAGCAGCGACTGCA 3' SEQ ID NO:32; 5' CGAGGTGCTGCGCCTGCG 3' SEQ ID NO:33; 5'CGAGGTGCTGCGCCTGTG 3' SEQ ID NO:34; 5 5'GGGATCACATCGTGGAGATG 3' SEQ ID NO:35; and 5'GGGATCACAACGAGGAGAAG 3' SEQ ID NO:36.

12. The method according to claim 1 10 including the further steps of forming said treated sample by (a) admixing a sample to be assayed with a plurality of nucleic acid probes to form a hybridization composition, wherein the 3'-terminal 15 region of each of said nucleic acid probes (i) hybridize with partial or total complementarity to said nucleic acid target sequence when that sequence is present in the sample and (ii) include an identifier nucleotide; 20 (b) maintaining said hybridization composition for a time period sufficient to form a treated sample that may contain said predetermined nucleic acid target sequences hybridized with their respective nucleic acid probes. 25

13. The method according to claim 1 wherein nucleic acid sample is obtained from a biological sample. WO 00/49181 PCT/USOO/04243 -150

14. The method according to claim 13 wherein a predetermined nucleic acid target sequence is a microbial or viral nucleic acid. 5

15. A method for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample that comprises the steps of: (A) admixing a sample to be assayed with a 10 plurality of nucleic acid probes to form a hybridization composition, wherein the 3'-terminal region of each of said nucleic acid probes (i) hybridizes with partial or total complementarity to at least one said predetermined nucleic acid target 15 sequence when that sequence is present in the sample and (ii) includes an identifier nucleotide; (B) maintaining said hybridization composition for a time period sufficient to form a treated sample that may contain said predetermined 20 nucleic acid target sequence hybridized with a nucleic acid probe; (C) admixing the treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3' 25 terminus of a hybridized nucleic acid probe to form a treated reaction mixture; (D) maintaining the treated reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and 30 release identifier nucleotides therefrom; and WO 00/49181 PCT/USOO/04243 -151 (E) analyzing for the presence of released identifier nucleotides to obtain an analytical output, the analytical output indicating the presence or absence of at least one of said plurality of 5 nucleic acid target sequences.

16. The method according to claim 15 wherein said analytical output is obtained by luminescence spectroscopy. 10

17. The method according to claim 15 wherein said analytical output is obtained by fluorescence spectroscopy. 15

18. The method according to claim 15 wherein said analytical output is obtained by mass spectrometry.

19. The method according to claim 15 20 wherein said analytical output is obtained by absorbance spectroscopy.

20. The method according to claim 15 wherein the analytical output obtained when one of 25 said nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is greater than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid 30 target sequences. WO 00/49181 PCT/USOO/04243 -152

21. The method according to claim 15 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence 5 is less than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences. 10

22. The method according to claim 15 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with total complementarity to one nucleic acid target sequence is greater than the analytical output when all of the 15 nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences.

23. The method according to claim 15 20 wherein the analytical output obtained when one of said nucleic acid probes hybridize with total complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with partial 25 complementarity to their respective nucleic acid target sequences.

24. The method according to claim 15 wherein said enzyme whose activity is to release 30 nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes WO 00/49181 PCT/USOO/04243 -153 hybridized nucleic acids whose bases in the 3' terminal region are matched with total complementarity. 5

25. The method according to claim 15 wherein said enzyme whose activity is to release nucleotides exhibits a 3'->5'-exonuclease activity, depolymerizing hybridized nucleic acids having one or more mismatched bases in the 3'-terminal region of 10 the hybridized probe.

26. The method according to claim 15 wherein said nucleic acid probes comprise sequences complementary to nucleic acid sequences associated 15 with blood coagulation.

27. The method according to claim 26 wherein said nucleic acid sequences associated with blood coagulation comprise 20 (a) a sequence of at least ten nucleotides of the Factor V Leiden mutation; and (b) a sequence of at least ten nucleotides of prothrombin. 25

28. The method according to claim 27 wherein said nucleic acid sequences associated with blood coagulation is selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ 30 ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, WO 00/49181 PCT/USOO/04243 -154 SEQ ID NO:47, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46.

29. The method according to claim 15 5 wherein said nucleic acid probes comprise sequences complementary to nucleic acid sequences associated with cystic fibrosis.

30. The method according to claim 29 10 wherein said nucleic acid probes comprise sequences complementary to nucleic acid sequences associated with the cystic fibrosis delta F508 mutation.

31. The method according to claim 29 15 wherein said nucleic acid probes are SEQ ID NO:95 or SEQ ID NO:96.

32. A method for determining the presence or absence of a specific base in a nucleic acid 20 target sequence in a sample to be assayed that comprises the steps of: (A) admixing a sample to be assayed with a plurality of nucleic acid probes to form a hybridization composition, wherein the 3'-terminal 25 region of at least one of said nucleic acid probes (i) is substantially complementary to said nucleic acid target sequence and comprises at least one predetermined nucleotide at an interrogation position, and (ii) includes an identifier nucleotide, 30 and wherein said nucleic acid target sequence WO 00/49181 PCT/USOO/04243 -155 comprises at least one specific base whose presence or absence is to be determined; (B) maintaining said hybridization composition for a time period sufficient to form a 5 treated sample, wherein said interrogation position of the probe is a nucleotide that is aligned with said specific base to be identified in said target sequence, when present, so that base pairing can occur; 10 (C) admixing the treated sample with an enzyme whose activity is to release one or more nucleotides from the 3'-terminus of a hybridized nucleic acid probe to depolymerize the hybrid and form a treated reaction mixture; 15 (D) maintaining the treated reaction mixture for a time period sufficient to release an identifier nucleotide therefrom; and (E) analyzing for the presence or absence of released identifier nucleotide to obtain an 20 analytical output that indicates the presence or absence of said specific base to be identified.

33. The method according to claim 32 wherein the identifier nucleotide is at the 25 interrogation position.

34. The method according to claim 32 wherein said analytical output is obtained by luminescence spectroscopy. 30 WO 00/49181 PCT/USOO/04243 -156

35. The method according to claim 32 wherein said analytical output is obtained by fluorescence spectroscopy. 5

36. The method according to claim 32 wherein said analytical output is obtained by mass spectrometry.

37. The method according to claim 32 10 wherein said nucleic acid target sequence is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid.

38. The method according to claim 37, 15 further comprising a first probe, a second probe, a third probe and a fourth probe.

39. The method according to claim 38 wherein said interrogation position of said first 20 probe comprises a nucleic acid residue that is a deoxyadenosine or adenosine residue, said interrogation position of said second probe comprises a nucleic acid residue that is a deoxythymidine or uridine residue, said interrogation position of said 25 third probe comprises a nucleic acid residue that is a deoxyguanosine or guanosine residue, and said fourth nucleic acid probe comprises a nucleic acid residue that is a deoxycytosine or cytosine residue. 30

40. The method according to claim 32 wherein the analytical output obtained when one of WO 00/49181 PCT/US0O/04243 -157 said nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is greater than the analytical output when all of the nucleic acid probes hybridize with total 5 complementarity to their respective nucleic acid target sequences.

41. The method according to claim 32 wherein the analytical output obtained when one of 10 said nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid 15 target sequences.

42. The method according to claim 32 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with total 20 complementarity to one nucleic acid target sequence is greater than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences. 25

43. The method according to claim 32 wherein the analytical output obtained when one of said nucleic acid probes hybridize with total complementarity to one target nucleic acid sequence 30 is less than the analytical output when all of the nucleic acid probes hybridize with partial WO 00/49181 PCT/US0O/04243 -158 complementarity to their respective nucleic acid target sequences.

44. The method according to claim 32 5 wherein said enzyme whose activity is to release nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic acids whose bases in the 3'terminal region of the probe are matched with total 10 complementarity.

45. The method according to claim 32 wherein said enzyme whose activity is to release nucleotides exhibits a 31 to 5' exonuclease activity, 15 depolymerizing hybridized nucleic acids having one or more mismatched bases at the 3'-terminus of the hybridized probe.

46. A method for determining the presence 20 or absence of a plurality of first nucleic acid targets in a nucleic acid sample containing those targets or a plurality of substantially identical second targets that comprises the steps of: (A) admixing said sample to be assayed with 25 one or more nucleic acid probes to form a hybridization composition, wherein said first and second nucleic acid targets comprise a region of sequence identity except for at least a single nucleotide at a predetermined position that differs 30 between the targets, and wherein said nucleic acid probe (i) is substantially complementary to said WO 00/49181 PCT/USOO/04243 -159 nucleic acid target region of sequence identity and comprises at least one nucleotide at an interrogation position, said interrogation position of the probe being aligned with said predetermined position of a 5 target when a target and probe are hybridized and (ii) includes an identifier nucleotide in the 3' terminal region; (B) maintaining said hybridization composition for a time period sufficient to form a 10 treated sample wherein the nucleotide at said interrogation position of said probe is aligned with the nucleotide at said predetermined position of said target in said region of identity; (C) admixing the treated sample with a 15 depolymerizing amount an enzyme whose activity is to release one or more nucleotides from the 3'-terminus of a hybridized nucleic acid probe to form a treated reaction mixture; (D) maintaining the treated reaction 20 mixture for a time period sufficient to release identifier nucleotide and depolymerize said hybridized nucleic acid probe; and (E) analyzing for the presence of released identifier nucleotide to obtain an analytical output, 25 said analytical output indicating the presence or absence of said nucleotide at said predetermined region and thereby the presence or absence of a first or second nucleic acid target. WO 00/49181 PCT/US00/04243 -160

47. The method according to claim 46 wherein said analytical output is obtained by fluorescence spectroscopy. 5

48. The method according to claim 46 wherein said analytical output is obtained by mass spectrometry.

49. The method according to claim 46 10 wherein said nucleic acid target sequence is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid.

50. The method according to claim 46 15 wherein said first probe comprises a nucleotide at said interrogation position that is complementary to a first target nucleic acid at said predetermined position, and said second probe comprises a nucleotide at the interrogation position that is 20 complementary to a second target nucleic acid at said predetermined position.

51. The method according to claim 46 wherein the analytical output obtained when one of 25 said nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is greater than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid 30 target sequences. WO 00/49181 PCT/USOO/04243 -161

52. The method according to claim 46 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence 5 is less than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences. 10

53. The method according to claim 46 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with total complementarity to one nucleic acid target sequence is greater than the analytical output when all of the 15 nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences.

54. The method according to claim 46 20 wherein the analytical output obtained when one of said nucleic acid probes hybridizes with total complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with partial 25 complementarity to their respective nucleic acid target sequences.

55. The method according to claim 46 wherein said enzyme whose activity is to release 30 nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes WO 00/49181 PCT/USOO/04243 -162 hybridized nucleic acids whose bases in the 3' terminal region are matched with total complementarity. 5

56. The method according to claim 46 wherein said enzyme whose activity is to release nucleotides exhibits a 3'-+5'-exonuclease activity, depolymerizing hybridized nucleic acids having one or more mismatched bases in the 3'-terminal region of 10 the hybridized probe.

57. A kit for determining the presence or absence of a plurality of predetermined nucleic acid target sequence in a nucleic acid sample comprising: 15 (A) an enzyme whose activity is to release one or more nucleotides from the 3' terminus of a hybridized nucleic acid probe; and (B) a plurality of nucleic acid probes, each of said nucleic acid probes being complementary 20 to nucleic acid target sequence.

58. A kit for determining the presence or absence of a plurality of predetermined nucleic acid target sequence in a nucleic acid sample comprising: 25 (A) an enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotide as a nucleoside triphosphate from hybridized nucleic acid probe; (B) pyrophosphate; WO 00/49181 PCT/USOO/04243 -163 (C) a plurality of nucleic acid probes, each of said nucleic acid probes being complementary to said predetermined nucleic acid target sequence. 5

59. The kit according to claim 58 wherein said nucleic acid probes comprise sequences complementary to nucleic acid sequences associated with blood coagulation. 10

60. The kit according to claim 59 wherein said nucleic acid sequences associated with blood coagulation comprise (a) a sequence of at least ten nucleotides of the Factor V Leiden mutation; and 15 (b) a sequence of at least ten nucleotides of prothrombin.

61. The kit according to claim 60 wherein said nucleic acid sequences associated with blood 20 coagulation is selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46. 25

62. The kit according to claim 58 wherein said nucleic acid probes comprise sequences complementary to nucleic acid sequences associated with cystic fibrosis. 30 WO 00/49181 PCT/USOO/04243 -164

63. The kit according to claim 62 wherein said nucleic acid probes comprise sequences complementary to nucleic acid sequences associated 5 with the cystic fibrosis delta F508 mutation.

64. The kit according to claim 63 wherein said nucleic acid probes are SEQ ID NO:95 or SEQ ID NO:96. 10

65. A kit for determining the presence or absence of a plurality of predetermined nucleic acid target sequence in a nucleic acid sample comprising: (A) an enzyme whose activity is to release one or more nucleotides from the 3' terminus of a 15 hybridized nucleic acid probe; and (B) instructions for use.

66. A composition for determining the presence or absence of a plurality of predetermined 20 nucleic acid target sequences in a nucleic acid sample comprising an aqueous solution that contains: (A) a purified and isolated enzyme whose activity is to release one or more nucleotides from the 3' terminus of a hybridized nucleic acid probe; 25 and (B) a plurality of nucleic acid probes, each of said nucleic acid probes being complementary to said predetermined nucleic acid target sequence. 30

67. A method for determining the presence or absence of a plurality of nucleic acid target WO 00/49181 PCT/USOO/04243 -165 sequences, each containing an interrogation position, in a nucleic acid sample that comprises the steps of: (A) providing a treated sample that contains a nucleic acid sample that may include said 5 nucleic acid target sequences hybridized with their respective nucleic acid probes, each probe being comprised of three sections, (i) a first section that contains the probe 3'-terminal about 10 to about 30 nucleotides that are complementary to its nucleic 10 acid target sequence at positions beginning about 1 to about 30 nucleic acids downstream of said interrogation position of the target sequence, (ii) a 5'-terminal region of about 10 to about 200 nucleic acids in length and having the identical sequence of 15 said nucleic acid target sequence, and (iii) an optional third section that contains zero to about 50 nucleic acids that are not complementary to said nucleic acid sample; (B) extending said nucleic acid probes in 20 a 3' direction to form second probes hybridized to the nucleic acid sample as second hybrids; (D) denaturing said second hybrids to separate said second probes from said nucleic acid target sequences; 25 (E) renaturing said aqueous composition to form hairpin structures from said second probes; (F) admixing the hairpin structure containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more 30 nucleotides from the 3'-terminus of a nucleic acid hybrid to form a treated reaction mixture; WO 00/49181 PCT/USOO/04243 -166 (G) maintaining the treated reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release one or more nucleotides from the 3'-terminus 5 therefrom; and (H) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said nucleic acid target sequences. 10

68. A method for determining the presence or absence of a plurality of nucleic acid target sequences, or a specific base within the target sequences, in a nucleic acid sample, that comprises 15 the steps of: (A) providing a treated sample that contains a nucleic acid sample that may include a plurality of nucleic acid target sequences hybridized with their respective first nucleic acid probes as a 20 first hybrid, said first probes each being comprised of at least two sections, a first section containing the probe 3'-terminal about 10 to about 30 nucleotides that are complementary to the target nucleic acid sequence at a position beginning about 5 25 to about 30 nucleotides downstream of the target interrogation position, a second section of the first probe containing about 5 to about 30 nucleotides that are a repeat of the target sequence from the interrogation position to about 10 to about 30 30 nucleotides downstream of the interrogation position that does not hybridize to said first section of the WO 00/49181 PCT/USOO/04243 -167 probe, and an optional third section of the probe located between the first and second sections of the probe that is zero to about 50 nucleotides in length and comprises a sequence that does not hybridize to 5 either the first or second section of the probe; (B) extending the first hybrid in the treated sample at the 3' -end of the first probes, thereby extending the first probes past the interrogation position and forming an extended first 10 hybrid that includes an interrogation position; (C) denaturing an aqueous composition of the extended first hybrid to separate the two nucleic acid strands and form an aqueous composition containing separated target nucleic acids and 15 separated extended first probes; (D) annealing to each of the extended first probes second probes that are about 10 to about 30 nucleotides in length and are complementary to the extended first probes at a position beginning about 5 20 to about 2000 nucleotides downstream of the interrogation position in the extended first probes, thereby forming a second hybrid; (E) extending the second hybrid at the 3' end of the second probes until that extension reaches 25 the 5'-end of the extended first probes, thereby forming a second extended hybrid containing a second extended probe whose 3'-region includes an identifier nucleotide; (F) denaturing an aqueous composition of 30 the extended second hybrid to separate the nucleic acid strands and form an aqueous composition WO 00/49181 PCT/USOO/04243 -168 containing separated extended first and second probes; (G) cooling the aqueous composition to form a hairpin structure from the separated extended 5 second probes to form a hairpin structure-containing composition; (H) admixing the hairpin structure containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more 10 nucleotides from the 3'-terminus of a nucleic acid hybrid to form a treated reaction mixture; (I) maintaining the reaction mixture for a time period sufficient to release 3'-terminal region identifier nucleotides; and 15 (J) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said predetermined nucleic acid target sequence or a specific base within the target 20 sequence.

69. The method according to claim 68 wherein said analytical output is obtained by luminescence spectroscopy. 25

70. The method according to claim 68 wherein said analytical output is obtained by fluorescence spectroscopy. WO 00/49181 PCT/USOO/04243 -169

71. The method according to claim 68 wherein said analytical output is obtained by mass spectrometry. 5

72. The method according to claim 68 wherein said analytical output is obtained by absorbance spectroscopy.

73. A method for determining the presence 10 or absence of a plurality of nucleic acid target sequences containing an interrogation position in a nucleic acid sample that comprises the steps of: (A) providing a treated sample that contains a nucleic acid sample that may include said plurality 15 of nucleic acid target sequences, each hybridized with its respective nucleic acid probe that is comprised of three sections, (i) a first section that contains the probe 3'-terminal about 10 to about 30 nucleotides that are complementary to the nucleic 20 acid target sequence at positions beginning about 1 to about 30 nucleic acids downstream of said interrogation position of the target sequence, (ii) a 5'-terminal region of about 10 to about 200 nucleic acids in length and having the identical sequence of 25 said nucleic acid target sequence, and (iii) an optional third section that contains zero to about 50 nucleic acids that are not complementary to said nucleic acid sample, and ; (B) extending said nucleic acid probes in a 3' 30 direction to form second probes hybridized to the nucleic acid sample as a second hybrid; WO 00/49181 PCT/USOO/04243 -170 (D) denaturing said second hybrid to separate said second probes from said nucleic acid target sequences; (E) renaturing said aqueous composition to form 5 hairpin structures from said second probes; (F) admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3'-terminus of a nucleic acid hybrid to form 10 a treated reaction mixture; (G) maintaining the treated reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release one or more nucleotides from the 3'-terminus therefrom; 15 and (H) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said nucleic acid target sequences. 20

74. The method according to claim 73 wherein said analytical output is obtained by luminescence spectroscopy. 25

75. The method according to claim 73 wherein said analytical output is obtained by fluorescence spectroscopy.

76. The method according to claim 73 wherein 30 said analytical output is obtained by mass spectrometry. WO 00/49181 PCT/USOO/04243 -171

77. The method according to claim 73 wherein said analytical output is obtained by absorbance spectroscopy. 5

78. The method according to claim 73 wherein the analytical output is distinguishable for the different nucleic acid target sequences. 10

79. A method for determining the presence or absence of a plurality of nucleic acid target sequences, or a specific base within a target sequence, in a nucleic acid sample, that comprises the steps of: 15 (A) providing a treated sample that contains a nucleic acid sample that may include a plurality of nucleic acid target sequences, each hybridized with its respective first nucleic acid probe as a first hybrid, said first probes being comprised of at least 20 two sections, a first section containing the probe 3'-terminal about 10 to about 30 nucleotides that are complementary to the target nucleic acid sequence at a position beginning about 5 to about 30 nucleotides downstream of the target interrogation position, a 25 second section of the first probe containing about 5 to about 30 nucleotides that are a repeat of the target sequence from the interrogation position to about 10 to about 30 nucleotides downstream of the interrogation position that does not hybridize to 30 said first section of the probe, and an optional third section of the probe located between the first WO 00/49181 PCT/USOO/04243 -172 and second sections of the probe that is zero to about 50 nucleotides in length and comprises a sequence that does not hybridize to either the first or second section of the probe; 5 (B) extending the first hybrid in the treated sample at the 3'-end of the first probes, thereby extending the first probes past the interrogation position and forming an extended first hybrid that includes an interrogation position; 10 (C) denaturing an aqueous composition of the extended first hybrid to separate the two nucleic acid strands and form an aqueous composition containing separated target nucleic acids and a separated extended first probes; 15 (D) annealing to the extended first probes a second probe that is about 10 to about 30 nucleotides in length and is complementary to the extended first probe at a position beginning about 5 to about 2000 nucleotides downstream of the interrogation position 20 in the extended first probes, thereby forming a second hybrid; (E) extending the second hybrid at the 3'-end of the second probes until that extension reaches the 5'-end of the extended first probe, thereby forming a 25 second extended hybrid containing a second extended probe whose 3'-region includes an identifier nucleotide; (F) denaturing an aqueous composition of the extended second hybrid to separate the two nucleic 30 acid strands and form an aqueous composition WO 00/49181 PCT/USOO/04243 -173 containing separated extended first and second probes; (G) cooling the aqueous composition to form a hairpin structure from the separated extended second 5 probe to form a hairpin structure-containing composition; (H) admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides 10 from the 3'-terminus of a nucleic acid hybrid to form a treated reaction mixture; (I) maintaining the reaction mixture for a time period sufficient to release 3'-terminal region identifier nucleotides; and 15 (J) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said predetermined nucleic acid target sequences or a specific base within a target 20 sequence.

80. The method according to claim 79 wherein said analytical output is obtained by luminescence spectroscopy. 25

81. The method according to claim 79 wherein said analytical output is obtained by fluorescence spectroscopy. WO 00/49181 PCT/US0O/04243 -174

82. The method according to claim 79 wherein said analytical output is obtained by mass spectrometry. 5

83. The method according to claim 79 wherein said analytical output is obtained by absorbance spectroscopy.

84. The method according to claim 79 wherein 10 said analytical output is distinguishable for the various predetermined nucleic acid target sequences.

85. A kit for determining the presence or absence of a plurality of predetermined nucleic acid 15 target sequences in a nucleic acid sample comprising: (A) a purified and isolated enzyme whose activity is to release one or more nucleotides from the 3' terminus of a hybridized nucleic acid probe; and 20 (B) a plurality of nucleic acid probes, each of said nucleic acid probes being complementary to a nucleic acid target sequence.

86. The kit according to claim 85 wherein the 25 predetermined nucleic acid target sequences are associated with blood coagulation.

87. The kit according to claim 86 wherein the nucleic acid probes comprise a plurality of the 30 following nucleic acid sequences or their complementary sequences: WO 00/49181 PCT/USOO/04243 -175 5' CTGCTGCCCTCTGTATTCCTCG 3' SEQ ID NO:14; 5' CTGCTGCCCTCTGTATTCCTTG 3' SEQ ID NO:15; 5' GTGACTCTCAGCG 3' SEQ ID NO:87; 5' GTGACTCTCAGCA 3' SEQ ID NO:88; 5 5' GTGATTCTCAGCG 3' SEQ ID NO:89; 5' GTGATTCTCAGCA 3' SEQ ID NO:90; 5' GACAAAATACCTGTATTCCTCG 3' SEQ ID NO:91; 5' GACAAAATACCTGTATTCCTTG 3' SEQ ID NO:92; 5' GGAGCATTGAGGCTCG 3' SEQ ID NO:93; 10 5' GGAGCATTGAGGCTTG 3' SEQ ID NO:94; 5' GACAAAATACCTGTATTCCTTG 3' SEQ ID NO:47; 5' GTGATTCTCAGCA 3' SEQ ID NO:44; 5' GTGATTCTCAGCG 3' SEQ ID NO:45; and 5' GACAAAATACCTGTATTCCTCG 3' SEQ ID NO:46. 15

88. The kit according to claim 87 wherein the predetermined nucleic acid target sequences are useful for speciation. 20

89. The kit according to claim 88 wherein the nucleic acid probes comprise a plurality of the following nucleic acid sequences or their complementary sequences: 5' CCAGACGCCTCA 3' SEQ ID NO:50; 25 5' ACCTTCACGCCA 3' SEQ ID NO:51; 5' TGCCGAGACGT 3' SEQ ID NO:52; 5' GCAGACACATCC 3' SEQ ID NO:53; 5' GGAATCTCCACG 3' SEQ ID NO:54; 5' ACATACACGCAA 3' SEQ ID NO:55; and 30 5' ATATGCACGCAA 3' SEQ ID NO:56. WO 00/49181 PCT/USO0/04243 -176

90. The kit according to claim 85 wherein the predetermined nucleic acid target sequences are associated with congenital adrenal hyperplasia. 5

91. The kit according to claim 90 wherein the nucleic acid probes comprise a plurality of the following nucleic acid sequences or their complementary sequences: 5' CGGAGCCTCCACCTCCCG SEQ ID NO:23; 10 5' CACCCTCCAGCCCCCAGC 3' SEQ ID NO:24; 5' CGGAGCCTCCACCTCCTG 3' SEQ ID NO:25; 5' CCTCACCTGCAGCATCAAC 3' SEQ ID NO:26; 5' CACCCTCCAGCCCCCAAC 3' SEQ ID NO:27; 5' CCTCACCTGCAGCATCATC 3' SEQ ID NO:28; 15 5' CCTGGAAGGGCACTT 3' SEQ ID NO:29; 5' CCTGGAAGGGCACGT 3' SEQ ID NO:30; 5' GATTCAGCAGCGACTGTA 3' SEQ ID NO:31; 5' GATTCAGCAGCGACTGCA 3' SEQ ID NO:32; 5' CGAGGTGCTGCGCCTGCG 3' SEQ ID NO:33; 20 5'CGAGGTGCTGCGCCTGTG 3' SEQ ID NO:34; 5'GGGATCACATCGTGGAGATG 3' SEQ ID NO:35; and 5'GGGATCACAACGAGGAGAAG 3' SEQ ID NO:36.

92. The kit according to claim 85 wherein said 25 nucleic acid probes comprise a fluorescent label.

93. The kit according to claim 85 wherein said nucleic acid probes comprise a non-natural nucleotide analog. 30 WO 00/49181 PCT/USOO/04243 -177

94. The kit according to claim 85 further comprising pyrophosphate.

95. The kit according to claim 85 further 5 comprising a nucleotide diphosphate kinase.

96. The composition according to 95, wherein said nucleoside diphosphate kinase is that encoded by Pyrococcus furiosis. 10

97. The kit according to claim 95 further comprising PRPP synthase.

98. The kit according to claim 95 further 15 comprising ADP.

99. A composition for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample 20 comprising an aqueous solution that contains: (A) a purified and isolated enzyme whose activity is to release one or more nucleotides from the 3' terminus of a hybridized nucleic acid probe; and 25 (B) a plurality of nucleic acid probes, each of said nucleic acid probes being complementary to a predetermined nucleic acid target sequence.

100. A composition of matter for determining the 30 presence or absence of a plurality of predetermined WO 00/49181 PCT/USOO/04243 -178 nucleic acid target sequences in a nucleic acid sample comprising an aqueous solution that contains: (A) a purified and isolated enzyme whose activity in the presence of pyrophosphate is to 5 release identifier nucleotide as a nucleoside triphosphate from hybridized nucleic acid probe; (B) adenosine 5' diphosphate; (C) pyrophosphate; (D) a purified and isolated nucleoside 10 diphosphate kinase; and (E) a plurality of nucleic acid probes, each of said nucleic acid probe being complementary to its respective predetermined nucleic acid target sequence. 15

101. The composition of matter according to claim 100, wherein said purified and isolated enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotides is a thermostable 20 polymerase.

102. The composition of matter according to claim 101, wherein said purified and isolated nucleoside diphosphate kinase is that encoded by 25 Pyrococcus furiosis.