The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, where the method includes forming a cleavage structure by incubating a sample comprising a target nucleic acid sequence with a probe having a secondary structure that changes upon binding of the probe to the target nucleic acid sequence, and cleaving the cleavage structure with a nuclease to release a nucleic acid fragment to generate a signal, wherein generation of the signal is indicative of the presence of a target nucleic acid sequence in a sample. The invention also relates to a method of detecting or measuring a target nucleic acid sequence in a sample, where the method includes forming a cleavage structure by incubating a sample containing a target nucleic acid sequence with a probe having a secondary structure that changes upon binding of the probe to a target nucleic acid sequence and cleaving the cleavage structure with a nuclease to generate a cleaved nucleic acid fragment.

The invention relates in general to methods of detecting or measuring a target nucleic acid sequence.
The fidelity of DNA replication, recombination, and repair is essential for maintaining genome stability, and all of these processes depend on 5xe2x80x2xe2x86x923xe2x80x2 exonuclease enzymes which are present in all organisms. For DNA repair, these enzymes are required for damaged fragment excision and recombinational mismatch correction. For replication, these nucleases are critical for the efficient processing of Okazaki fragments during lagging strand DNA synthesis. In Escherichia coli, this latter activity is provided by DNA polymerase I (Poll); E. coli strains with inactivating mutations in the Poll 5xe2x80x2xe2x86x923xe2x80x2 exonuclease domain are not viable due to an inability to process Okazaki fragments. Eukaryotic DNA polymerases, however, lack an intrinsic 5xe2x80x2xe2x86x923xe2x80x2 exonuclease domain, and this critical activity is provided by the multifunctional, structure-specific metallonuclease FEN-1 (fivexe2x80x2 exonuclease-1 or flap endonuclease-1), which also acts as an endonuclease for 5xe2x80x2 DNA flaps (Reviewed in Hosfield et al., 1998a, Cell, 95:135).
Methods of detecting and/or measuring a nucleic acid wherein an enzyme produces a labeled nucleic acid fragment are known in the art.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 disclose a method of cleaving a target DNA molecule by incubating a 5xe2x80x2 labeled target DNA with a DNA polymerase isolated from Thermus aquaticus (Taq polymerase) and a partially complementary oligonucleotide capable of hybridizing to sequences at the desired point of cleavage. The partially complementary oligonucleotide directs the Taq polymerase to the target DNA through formation of a substrate structure containing a duplex with a 3xe2x80x2 extension opposite the desired site of cleavage wherein the non-complementary region of the oligonucleotide provides a 3xe2x80x2 arm and the unannealed 5xe2x80x2 region of the substrate molecule provides a 5xe2x80x2 arm. The partially complementary oligonucleotide includes a 3xe2x80x2 nucleotide extension capable of forming a short hairpin either when unhybridized or when hybridized to a target sequence at the desired point of cleavage. The release of labeled fragment is detected following cleavage by Taq polymerase.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 disclose the generation of mutant, thermostable DNA polymerases that have very little or no detectable synthetic activity, and wild type thermostable nuclease activity. The mutant polymerases are said to be useful because they lack 5xe2x80x2 to 3xe2x80x2 synthetic activity; thus synthetic activity is an undesirable side reaction in combination with a DNA cleavage step in a detection assay.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 disclose that wild type Taq polymerase or mutant Taq polymerases that lack synthetic activity can release a labeled fragment by cleaving a 5xe2x80x2 end labeled hairpin structure formed by heat denaturation followed by cooling, in the presence of a primer that binds to the 3xe2x80x2 arm of the hairpin structure. Further, U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 teach that the mutant Taq polymerases lacking synthetic activity can also cleave this hairpin structure in the absence of a primer that binds to the 3xe2x80x2 arm of the hairpin structure.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 also disclose that cleavage of this hairpin structure in the presence of a primer that binds to the 3xe2x80x2 arm of the hairpin structure by mutant Taq polymerases lacking synthetic activity yields a single species of labeled cleaved product, while wild type Taq polymerase produces multiple cleavage products and converts the hairpin structure to a double stranded form in the presence of dNTPs, due to the high level of synthetic activity of the wild type Taq enzyme.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 also disclose that mutant Taq polymerases exhibiting reduced synthetic activity, but not wild type Taq polymerase, can release a single labeled fragment by cleaving a linear nucleic acid substrate comprising a 5xe2x80x2 end labeled target nucleic acid and a complementary oligonucleotide wherein the complementary oligonucleotide hybridizes to a portion of the target nucleic acid such that 5xe2x80x2 and 3xe2x80x2 regions of the target nucleic acid are not annealed to the oligonucleotide and remain single stranded.
There is a need in the art for a method of generating a signal that can be easily distinguished from oligonucleotide fragments that may arise from nuclease contaminants, using a nucleic acid cleavage reaction.
There is a need in the art for a method of generating a signal that utilizes a probe comprising secondary structure wherein some or all of the self-complementary regions of the probe that anneal to form the secondary structure are melted when the probe hybridizes with a target nucleic acid, thereby reducing non-specific binding of the probe to the target, and increasing the specificity of the assay.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 also disclose a method of cleaving a labeled nucleic acid substrate at naturally occurring areas of secondary structure. According to this method, biotin labeled DNA substrates are prepared by PCR, mixed with wild type Taq polymerase or CleavaseBN (a mutant Taq polymerase with reduced synthetic activity and wild type 5xe2x80x2 to 3xe2x80x2 nuclease activity), incubated at 95xc2x0 C. for 5 seconds to denature the substrate and then quickly cooled to 65xc2x0 C. to allow the DNA to assume its unique secondary structure by allowing the formation of intra-strand hydrogen bonds between the complementary bases. The reaction mixture is incubated at 65xc2x0 C. to allow cleavage to occur and biotinylated cleavage products are detected.
There is a need in the art for a method of generating a signal using a nucleic acid cleavage reaction wherein the cleavage structure is not required to contain areas of secondary structure.
Methods of detecting and/or measuring a nucleic acid wherein a FEN-1 enzyme is used to generate a labeled nucleic acid fragment are known in the art.
U.S. Pat. No. 5,843,669 discloses a method of detecting polymorphisms by cleavase fragment length polymorphism analysis using a thermostable FEN-1 nuclease in the presence or absence of a mutant Taq polymerase exhibiting reduced synthetic activity. According to this method, double stranded Hepatitis C virus (HCV) DNA fragments are labeled by using 5xe2x80x2 end labeled primers (labeled with TMR fluorescent dye) in a PCR reaction. The TMR labeled PCR products are denatured by heating to 95xc2x0 C. and cooled to 55xc2x0 C. to generate a cleavage structure. U.S. Pat. No. 5,843,669 discloses that a cleavage structure comprises a region of a single stranded nucleic acid substrate containing secondary structure. Cleavage is carried out in the presence of CleavaseBN nuclease, FEN-1 nuclease derived from the archaebacteria Methanococcus jannaschii or both enzymes. Labeled reaction products are visualized by gel electrophoresis followed by fluoroimaging. U.S. Pat. No. 5,843,669 discloses that CleavaseBN nuclease and Methanococcus jannaschii FEN-1 nuclease produce cleavage patterns that are easily distinguished from each other, and that the cleavage patterns from a reaction containing both enzymes include elements of the patterns produced by cleavage with each individual enzyme but are not merely a composite of the cleavage patterns produced by each individual enzyme. This indicates that some of the fragments that are not cleaved by one enzyme (and which appear as a band in that enzyme""s pattern) can be cleaved by a second enzyme in the same reaction mixture.
Lyamichev et al. disclose a method for detecting DNAs wherein overlapping pairs of oligonucleotide probes that are partially complementary to a region of target DNA are mixed with the target DNA to form a 5xe2x80x2 flap region, and wherein cleavage of the labeled downstream probe by a thermostable FEN-1 nuclease produces a labeled cleavage product. Lyamichev et al. also disclose reaction conditions wherein multiple copies of the downstream oligonucleotide probe can be cleaved for a single target sequence in the absence of temperature cycling, so as to amplify the cleavage signal and allow quantitative detection of target DNA at sub-attomole levels (Lyamichev et al., 1999, Nat. Biotechnol., 17:292).
The polymerase chain reaction (PCR) technique, is disclosed in U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. A repetitive series of reaction steps involving template denaturation, primer annealing and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5xe2x80x2 ends of the primers. PCR is reported to be capable of producing a selective enrichment of a specific DNA sequence by a factor of 109. The PCR method is also described in Saiki et al., 1985, Science, 230:1350.
While the PCR technique is an extremely powerful method for amplifying nucleic acid sequences, the detection of the amplified material requires additional manipulation and subsequent handling of the PCR products to determine whether the target DNA is present. It is desirable to decrease the number of subsequent handling steps currently required for the detection of amplified material. An assay system, wherein a signal is generated while the target sequence is amplified, requires fewer handling steps for the detection of amplified material, as compared to a PCR method that does not generate a signal during the amplification step.
U.S. Pat. Nos. 5,210,015 and 5,487,972 disclose a PCR based assay for releasing labeled probe comprising generating a signal during the amplification step of a PCR reaction in the presence of a nucleic acid to be amplified, Taq polymerase that has 5xe2x80x2 to 3xe2x80x2 exonuclease activity and a 5xe2x80x2, 3xe2x80x2 or 5xe2x80x2 and 3xe2x80x2 end-labeled probe comprising a region complementary to the amplified region and an additional non-complementary 5xe2x80x2 tail region. U.S. Pat. Nos. 5,210,015 and 5,487,972 disclose further that this PCR based assay can liberate the 5xe2x80x2 labeled end of a hybridized probe when the Taq polymerase is positioned near the labeled probe by an upstream probe in a polymerization independent manner, e.g. in the absence of dNTPs.
There is a need in the art for a method of detecting or measuring a target nucleic acid sequence that does not require multiple steps.
There is also a need in the art for a PCR process for detecting or measuring a target nucleic acid sequence that does not require multiple steps subsequent to the amplification process.
There is also a need in the art for a PCR process for detecting or measuring a target nucleic acid sequence that allows for concurrent amplification and detection of a target nucleic acid sequence in a sample.
The invention provides a method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample comprising forming a cleavage structure by incubating a sample comprising a target nucleic acid sequence with a probe having a secondary structure that changes upon binding of the probe to a target nucleic acid sequence, and cleaving the cleavage structure with a nuclease to generate a signal, wherein the cleaving is performed at a cleaving temperature and the secondary structure of the probe when not bound to the target nucleic acid sequence is stable at or below the cleaving temperature and wherein generation of the signal is indicative of the presence of a target nucleic acid sequence in the sample.
As used herein, a xe2x80x9cprobexe2x80x9d refers to a single stranded nucleic acid comprising a region or regions that are complementary to a target nucleic acid sequence (e.g., target nucleic acid binding sequences, for example C in FIG. 4). A xe2x80x9cprobexe2x80x9d according to the invention has a secondary structure that changes upon binding of the probe to the target nucleic acid sequence. A xe2x80x9cprobexe2x80x9d according to the invention binds to a target nucleic acid sequence to form a cleavage structure that can be cleaved by a nuclease, wherein cleaving is performed at a cleaving temperature, and wherein the secondary structure of the probe when not bound to the target nucleic acid sequence is, preferably, stable at or below the cleaving temperature. A probe according to the invention cannot be cleaved to generate a signal by a xe2x80x9cnucleasexe2x80x9d, as defined herein, prior to binding to a target nucleic acid. In one embodiment of the invention, a probe may comprise a region that cannot bind or is not complementary to a target nucleic acid sequence. In another embodiment of the invention, a probe does not have a secondary structure when bound to a target nucleic acid.
As used herein, xe2x80x9csecondary structurexe2x80x9d refers to a three-dimensional conformation (for example a hairpin, a stem-loop structure, an internal loop, a bulge loop, a branched structure or a pseudoknot, FIGS. 1 and 3; multiple stem loop structures, cloverleaf type structures or any three dimensional structure. As used herein, xe2x80x9csecondary structurexe2x80x9d includes tertiary, quaternary etc . . . structure. A probe comprising such a three-dimensional structure binds to a target nucleic acid sequence to form a cleavage structure that can be cleaved by a nuclease at a cleaving temperature. The three dimensional structure of the probe when not bound to the target nucleic acid sequence is, preferably, stable at or below the cleaving temperature. xe2x80x9cSecondary structurexe2x80x9d as used herein, can mean a sequence comprising a first single-stranded sequence of bases (referred to herein as a xe2x80x9ccomplementary nucleic acid sequencexe2x80x9d (for example b in FIG. 4)) followed by a second complementary sequence either in the same molecule (for example bxe2x80x2 in FIG. 4), or in a second molecule comprising the probe, folds back on itself to generate an antiparallel duplex structure, wherein the single-stranded sequence and the complementary sequence (that is, the complementary nucleic acid sequences) anneal by the formation of hydrogen bonds. Oligonucleotide probes, as used in the present invention include oligonucleotides comprising secondary structure, including, but not limited to molecular beacons, safety pins (FIG. 9), scorpions (FIG. 10), and sunrise/amplifluor probes (FIG. 11), the details and structures of which are described below and in the corresponding figures.
As used herein, first and second xe2x80x9ccomplementaryxe2x80x9d nucleic acid sequences are complementary to each other and can anneal by the formation of hydrogen bonds between the complementary bases.
A secondary structure also refers to the conformation of a nucleic acid molecule comprising an affinity pair, defined herein, wherein the affinity pair reversibly associates as a result of attractive forces that exist between the pair of moieties comprising the affinity pair.
A xe2x80x9cprobexe2x80x9d according to the invention can be unimolecular. As used herein, a xe2x80x9cunimolecularxe2x80x9d probe comprises a single molecule that binds to a target nucleic acid sequence to form a cleavage structure that can be cleaved by a nuclease, wherein cleaving is performed at a cleaving temperature, and wherein the secondary structure of the xe2x80x9cunimolecularxe2x80x9d probe when not bound to the target nucleic acid sequence is, preferably, stable at or below the cleaving temperature. Unimolecular probes useful according to the invention include but are not limited to beacon probes, probes comprising a hairpin, stem-loop, internal loop, bulge loop or pseudoknot structure, scorpion probes and sunrise/amplifluor probes.
A xe2x80x9cprobexe2x80x9d according to the invention can be more than one molecule (e.g., bi-molecular or multi-molecular). At least one of the molecules comprising a bi-molecular or multi-molecular probe binds to a target nucleic acid sequence to form a cleavage structure that can be cleaved by a nuclease, wherein cleaving is performed at a cleaving temperature, and wherein the secondary structure of the molecule of the probe when not bound to the target nucleic acid sequence is, preferably, stable at or below the cleaving temperature. The molecules comprising the multimolecular probe associate with each other via intramolecular bonds (e.g., hydrogen bonds or covalent bonds). For example, a heterologous loop (see FIG. 1), or a cloverleaf structure wherein one or more loops of the cloverleaf structure comprises a distinct molecule, and wherein the molecules that associate to form the cloverleaf structure associate via intramolecular bonding (e.g., hydrogen bonding or covalent bonding), are examples of multimolecular probes useful according to the invention.
As used herein, a xe2x80x9cmoleculexe2x80x9d refers to a polynucleotide, and includes a polynucleotide further comprising an attached member or members of an affinity pair.
A xe2x80x9cprobexe2x80x9d or a xe2x80x9cmoleculexe2x80x9d comprising a probe is 5-250 nucleotides in length, ideally from 17-40 nucleotides in length, although probes or a molecule comprising a probe of different lengths are useful.
A xe2x80x9cprobexe2x80x9d according to the invention has a target nucleic acid binding sequence that is from about 7 to about 140 nucleotides, and preferably from 10 to about 140 nucleotides. A xe2x80x9cprobexe2x80x9d according to the invention comprises at least first and second complementary nucleic acid sequences or regions that are 3-25, preferably 4-15, and more preferably 5-11 nucleotides long. The first and second complementary nucleic acid sequences preferably have the same length. The invention provides for a probe wherein the first and second complementary nucleic acid sequences are both located upstream of the target nucleic acid binding site. Alternatively, the first and second complementary nucleic acid sequences can both be located downstream of the target nucleic acid binding site. In another embodiment, the invention provides for a probe wherein the first complementary nucleic acid sequence is upstream of the target nucleic acid binding site and the second complementary nucleic acid sequence is downstream of the target nucleic acid binding site. In another embodiment, the invention provides for a probe wherein the second complementary nucleic acid sequence is upstream of the target nucleic acid binding site and the first complementary nucleic acid sequence is downstream of the target nucleic acid binding site. The actual length will be chosen with reference to the target nucleic acid binding sequence such that the secondary structure of the probe is, preferably, stable when the probe is not bound to the target nucleic acid at the temperature at which cleavage of a cleavage structure comprising the probe bound to a target nucleic acid is performed. As the target nucleic acid binding sequence increases in size up to 100 nucleotides, the length of the complementary nucleic acid sequences may increase up to 15-25 nucleotides. For a target nucleic acid binding sequence greater than 100 nucleotides, the length of the complementary nucleic acid sequences are not increased further. If the probe is also an allele-discriminating probe, the lengths of the complementary nucleic acid sequences are more restricted, as is discussed below.
As used herein, the xe2x80x9ctarget nucleic acid binding sequencexe2x80x9d refers to the region of the probe that binds specifically to the target nucleic acid.
A xe2x80x9chairpin structurexe2x80x9d or a xe2x80x9cstemxe2x80x9d refers to a double-helical region formed by base pairing between adjacent, inverted, complementary sequences in a single strand of RNA or DNA.
A xe2x80x9cstem-loopxe2x80x9d structure refers to a hairpin structure, further comprising a loop of unpaired bases at one end.
As used herein, a probe with xe2x80x9cstablexe2x80x9d secondary structure when not bound to a target nucleic acid sequence, refers to a secondary structure wherein more than 50% (e.g., 51%, 55%, 75% or 100%) of the base pairs that constitute the probe are not dissociated under conditions which permit hybridization of the probe to the target nucleic acid, but in the absence of the target nucleic acid.
xe2x80x9cStabilityxe2x80x9d of a nucleic acid duplex is determined by the melting temperature, or xe2x80x9cTmxe2x80x9d. The Tm of a particular nucleic acid duplex under specified conditions (e.g., salt concentration and/or the presence or absence of organic solvents) is the temperature at which half (50%) of the base pairs of the duplex molecule have disassociated (that is, are not hybridized to each other in a base-pair).
The xe2x80x9cstabilityxe2x80x9d of the secondary structure of a probe when not bound to the target nucleic acid is defined in a melting temperature assay, in a fluorescence resonance energy transfer (FRET) assay or a fluorescence quenching assay, (the details or which are described in a section entitled, xe2x80x9cDetermining the Stability or the Secondary Structure of a Probexe2x80x9d).
A probe useful in the invention preferably will have secondary structure that is xe2x80x9cstablexe2x80x9d, when not bound to target, at or below the temperature of the cleavage reaction. Thus, the temperature at which nuclease cleavage of a probe/target nucleic acid hybrid is performed according to the invention, must be lower than the Tm of the secondary structure. The secondary structure of the probe is xe2x80x9cstablexe2x80x9d in a melting temperature assay, at a temperature that is at or below the temperature of the cleavage reaction (i.e., at which cleavage is performed) if the level of light absorbance at the temperature at or below the temperature of the cleavage reaction is less than (i.e., at least 5% less than, preferably 20% less than and most preferably 25% less than, etc . . . ) the level of light absorbance at a temperature that is equal to or greater than the Tm of the probe (see FIGS. 12c and 12d).
According to the method of the invention, the stability of a secondary structure can be measured by a FRET assay or a fluorescence quenching assay (described in the section entitled, xe2x80x9cDetermining the Stability of the Secondary Structure of a Probexe2x80x9d). As used herein, a fluorescence quenching assay can include a FRET assay. A probe according to the invention is labeled with an appropriate pair of interactive labels (e.g., a FRET pair (for example as described in the section entitled, xe2x80x9cDetermining the Stability of the Secondary Structure of the Probexe2x80x9d, below) that can interact over a distance of, for example 2 nucleotides, or a non-FRET-pair, (e.g., tetramethylrhodamine and DABCYL) that can interact over a distance of, for example, 20 nucleotides. For example, a probe according to the invention may be labeled with a fluorophore and a quencher and fluorescence is then measured, in the absence of a target nucleic acid, at different temperatures. The Tm is the temperature at which the level of fluorescence is 50% of the maximal level of fluorescence observed for a particular probe, see FIG. 12e. The Tm for a particular probe wherein the nucleic acid sequence of the probe is known, can be predicted according to methods known in the art. Thus, fluorescence is measured over a range of temperatures, e.g., wherein the lower temperature limit of the range is at least 50xc2x0 Celsius below, and the upper temperature limit of the range is at least 50xc2x0 Celsius above the Tm or predicted Tm, for a probe according to the invention.
A secondary structure is herein defined as xe2x80x9cstablexe2x80x9d in a FRET assay at a temperature that is at or below the cleaving temperature if the level or wavelength of fluorescence is increased or decreased (e.g., at least 5%, preferably 20% and more preferably 25%, etc . . . ) as compared with the level or wavelength of FRET that is observed at the Tm of the probe (see FIGS. 12e and f). For example, an increase or a decrease in FRET can occur in a FRET assay according to the invention. In another embodiment, a shift in wavelength, which results in an increase in the new, shifted wavelength or, a decrease in the new shifted wavelength, can occur in a FRET assay according to the invention.
A xe2x80x9cchangexe2x80x9d in a secondary structure, according to the invention can be measured in a fluorescence quenching assay wherein a probe according to the invention comprises a fluorophore and a quencher that are positioned such that in the absence of a target nucleic acid, and at temperatures below the Tm of the probe there is quenching of the fluorescence (as described above). As used herein, a xe2x80x9cchangexe2x80x9d in secondary structure that occurs when a probe according to the invention binds to a target nucleic acid, refers to an increase in fluorescence in such an assay, such that the level of fluorescence after binding of the probe to the target nucleic acid at a temperature below the Tm of the probe, is greater than (e.g., at least 5%, preferably 5-20% and more preferably 25% or more) the level of fluorescence observed in the absence of a target nucleic acid sequence (see FIG. 12g).
A secondary structure, according to the invention, can be detected by subjecting a probe comprising a fluorophore and a quencher to a fluorescence quenching assay (as described above). A probe that exhibits a change in fluorescence that correlates with a change in temperature, see FIG. 12e, (e.g., fluorescence increases as the temperature of the reaction is increased) may be capable of forming a secondary structure.
As used herein, a xe2x80x9ccleaving temperaturexe2x80x9d that is useful according to the invention is a temperature that is less than (at least 5xc2x0 and preferably 10xc2x0) the Tm of a probe having a secondary structure. The xe2x80x9ccleaving temperaturexe2x80x9d is initially selected to be possible and preferably optimal for the particular nuclease being employed in the cleavage reaction.
Generally the 3xe2x80x2 terminus of the probe will be xe2x80x9cblockedxe2x80x9d to prohibit incorporation of the probe into a primer extension product. xe2x80x9cBlockingxe2x80x9d can be achieved by using non-complementary bases or by adding a chemical moiety such as biotin or a phosphate group to the 3xe2x80x2 hydroxl of the last nucleotide. Blocking can also be achieved by removing the 3xe2x80x2-OH or by using a nucleotide that lacks a 3xe2x80x2-OH such as dideoxynucleotide.
The term probe encompasses an allele-discriminating probe. As used herein, an xe2x80x9callele-discriminatingxe2x80x9d probe preferentially hybridizes to perfectly complementary target nucleic acid sequences and discriminates against sequences that vary by at least one nucleotide. A nucleic acid sequence which differs by at least one nucleotide, as compared to a target nucleic acid sequence, hereafter referred to as a xe2x80x9ctarget-like nucleic acid sequencexe2x80x9d, is thus not a target nucleic acid sequence for an allele-discriminating probe according to the invention.
Allele-discriminating probes do not hybridize sufficiently to a target-like nucleic acid sequence that contains one or more nucleotide mismatches as compared to the target nucleic acid complementary sequence and thus do not undergo a change in secondary structure upon binding to a target-like nucleic acid sequence in the presence of only a target-like nucleic acid sequence, and under conditions that would support hybridization of the allele discriminating probe to a target nucleic acid sequence.
In one embodiment, an xe2x80x9callele-discriminating probexe2x80x9d according to the invention refers to a probe that hybridizes to a target-like nucleic acid sequence that varies by at least one nucleotide from the target nucleic acid sequence, wherein the variant nucleotide(s) is/are not located in the allele-discriminating site. According to this embodiment of the invention, xe2x80x9can allele-discriminating probexe2x80x9d cannot bind to a target-like nucleic acid sequence that also varies by at least one nucleotide in the allele-discriminating site.
As used herein, xe2x80x9callele-discriminating sitexe2x80x9d refers to a region of a target nucleic acid sequence that is different (i.e., by at least one nucleotide) from the corresponding region in all possible alleles comprising the target nucleic acid sequence.
Allele-discriminating probes useful according to the invention also include probes that bind less effectively to a target-like sequence, as compared to a target sequence. The effectiveness of binding of a probe to a target sequence or a target-like sequence can be measured in a FRET assay, performed at a temperature that is below (at least 5xc2x0 and preferably 10xc2x0 or more) the Tm of the secondary structure of the probe, in the presence of a target-like sequence or a target sequence. The change in the level of fluorescence in the presence or absence of a target sequence compared to the change in the level of fluorescence in the presence or absence of a target-like sequence, provides an effective measure of the effectiveness of binding of a probe to a target or target-like sequence.
In a method according to the invention, a probe that binds less effectively to a target-like sequence as compared to a target sequence would undergo a smaller (e.g., preferably 25-50%, more preferably 50-75% and most preferably 75-90% of the value of the change in fluorescence upon binding to a target nucleic acid sequence) change in secondary structure, as determined by measuring fluorescence in a FRET or fluorescence quenching assay as described herein, upon hybridization to a target-like sequence as compared to a target nucleic acid sequence. In a method according to the invention, a probe that binds less effectively to a target-like sequence as compared to a target sequence would generate a signal that is indicative of the presence of a target-like nucleic acid sequence in a sample. However, the intensity of the signal would be altered (e.g., preferably 25-50%, more preferably 50-75% and most preferably 75-90% less than or more than the value of the change in fluorescence upon binding to a target nucleic acid sequence) the intensity of a signal generated in the presence of a target sequence, as described hereinabove for a smaller change.
A xe2x80x9csignal that is indicative of the presence of a xe2x80x9ctarget nucleic acid sequencexe2x80x9d or a xe2x80x9ctarget-like nucleic acid sequencexe2x80x9d refers to a signal that is equal to a signal generated from 1 molecule to 1020 molecules, more preferably about 100 molecules to 1017 molecules and most preferably about 1000 molecules to 1014 molecules of a target nucleic acid sequence or a target-like nucleic acid sequence.
As used herein, xe2x80x9caffinity pairxe2x80x9d refers to a pair of moieties (for example complementary nucleic acid sequences, protein-ligand, antibody-antigen, protein subunits, and nucleic acid binding proteins-binding sites) that can reversibly associate as a result of attractive forces that exist between the moieties.
In embodiments wherein the affinity pair comprises complementary nucleic acid regions that reversibly interact with one another, the lengths of the target nucleic acid binding sequences, and the nucleic acid sequences comprising the affinity pair, are chosen for the proper thermodynamic functioning of the probe under the conditions of the projected hybridization assay. Persons skilled in hybridization assays will understand that pertinent conditions include probe, target and solute concentrations, detection temperature, the presence of denaturants and volume excluders, and other hybridization-influencing factors. The length of a target nucleic acid binding sequence can range from 7 to about 140 nucleotides, preferably from 10 nucleotides to about 140 nucleotides. If the probe is also an allele-discriminating probe, the length is more restricted, as is discussed below.
In embodiments wherein the affinity pair comprises complementary nucleic acid regions that reversibly interact with one another, and cannot hybridize or are not complementary to a target nucleic acid sequence, the complementary nucleic acid region sequences of the affinity pair should be of sufficient length that under the conditions of the assay and at the detection temperature, when the probe is not bound to a target, the complementary nucleic acid sequences are associated. Depending upon the assay conditions used, complementary nucleic acid sequences of 3-25 nucleotides in length can perform this function. An intermediate range of 4-15, and more preferably 5-11, nucleotides is often appropriate. The actual length will be chosen with reference to the target nucleic acid binding sequence such that the secondary structure of the probe is stable when not bound to the target nucleic acid at the temperature at which cleavage of a cleavage structure comprising the probe bound to a target nucleic acid is performed. As the target nucleic acid binding sequence increases in size up to 100 nucleotides, the length of the complementary nucleic acid sequences may increase up to 15-25 nucleotides. For a target nucleic acid binding sequence greater than 100 nucleotides, the length of the complementary nucleic acid sequences are not increased further. If the probe is also an allele-discriminating probe, the lengths of the complementary nucleic acid sequences are more restricted, as is discussed below. Allele-discriminating probes that do not hybridize sufficiently to a target-like nucleic acid sequence that contain one or more nucleotide mismatches as compared to the target nucleic acid complementary sequence, must be designed such that, under the assay conditions used, reduction or elimination of secondary structure in the probe and hybridization with a target nucleic acid sequence will occur only when the target nucleic acid complementary sequence finds a perfectly complementary target sequence.
In one embodiment, an xe2x80x9callele-discriminating probexe2x80x9d according to the invention refers to a probe that hybridizes to a target-like nucleic acid sequence that varies by at least one nucleotide from the target nucleic acid sequence, wherein the variant nucleotide(s) is/are not located in the allele-discriminating site. According to this embodiment of the invention, xe2x80x9can allele-discriminating probexe2x80x9d cannot bind to a target-like nucleic acid sequence that also varies by at least one nucleotide in the allele-discriminating site.
In one embodiment of the invention, an allele discriminating probe according to the invention preferably comprises a target nucleic acid binding sequence from 6 to 50 and preferably from 7 to 25 nucleotides, and sequences of the complementary nucleic acid sequences from 3 to 8 nucleotides. The guanosine-cytidine content of the secondary structure and probe-target hybrids, salt, and assay temperature should all be considered, for example magnesium salts have a strong stabilizing effect that is particularly important to consider when designing short, allele-discriminating probes.
If an allele-discriminating probe is to have a target nucleic acid binding sequence near the upper limits of 50 nucleotides long, the sequence should be designed such that a single nucleotide mismatch to be discriminated against occurs at or near the middle of the target nucleic acid complementary sequence. For example, probes comprising a sequence that is 21 nucleotides long should preferably be designed so that the mismatch occurs opposite one of the 14 most centrally located nucleotides of the target nucleic acid complementary sequence and most preferably opposite one of the 7 most centrally located nucleotides. Designing a probe so that the mismatch to be discriminated against occurs in or near the middle of the target nucleic acid binding sequence/target-like nucleic acid binding sequence is believed to improve the performance of an allele-discriminating probe.
As used herein a xe2x80x9cnucleasexe2x80x9d or a xe2x80x9ccleavage agentxe2x80x9d refers to an enzyme that is specific for, that is, cleaves a cleavage structure according to the invention and is not specific for, that is, does not substantially cleave either a probe or a primer that is not hybridized to a target nucleic acid, or a target nucleic acid that is not hybridized to a probe or a primer. The term xe2x80x9cnucleasexe2x80x9d includes an enzyme that possesses 5xe2x80x2 endonucleolytic activity for example a DNA polymerase, e.g. DNA polymerase I from E. coli, and DNA polymerase from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Pyrococcus furiosus (Pfu) and Thermus flavus (Tfl). The term nuclease also embodies FEN nucleases. The term xe2x80x9cFEN nucleasexe2x80x9d encompasses an enzyme that possesses 5xe2x80x2 exonuclease and/or an endonuclease activity. The term xe2x80x9cFEN nucleasexe2x80x9d also embodies a 5xe2x80x2 flap-specific nuclease. A nuclease or cleavage agent according to the invention includes but is not limited to a FEN nuclease enzyme derived from Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus, human, mouse or Xenopus laevis. A nuclease according to the invention also includes Saccharomyces cerevisiae RAD27, and Schizosaccharomyces pombe RAD2, Pol I DNA polymerase associated 5xe2x80x2 to 3xe2x80x2 exonuclease domain, (e.g. E. coli, Thermus aquaticus (Taq), Thermus flavus (Tfl), Bacillus caldotenax (Bca), Streptococcus pneumoniae) and phage functional homologs of FEN including but not limited to T5 5xe2x80x2 to 3xe2x80x2 exonuclease, T7 gene 6 exonuclease and T3 gene 6 exonuclease. Preferably, only the 5xe2x80x2 to 3xe2x80x2 exonuclease domains of Taq, Tfl and Bca FEN nuclease are used. The term xe2x80x9cnucleasexe2x80x9d does not include RNAse H.
As used herein, xe2x80x9cwild typexe2x80x9d refers to a gene or gene product which has the characteristics of (i.e., either has the sequence of or encodes, for the gene, or possesses the sequence or activity of, for an enzyme) that gene or gene product when isolated from a naturally occurring source.
A xe2x80x9c5xe2x80x2 flap-specific nucleasexe2x80x9d (also referred to herein as a xe2x80x9cflap-specific nucleasexe2x80x9d) according to the invention is an endonuclease which can remove a single stranded flap that protrudes as a 5xe2x80x2 single strand. In one embodiment of the invention, a flap-specific nuclease according to the invention can also cleave a pseudo-Y structure. A substrate of a flap-specific nuclease according to the invention, comprises a target nucleic acid and an oligonucleotide probe, as defined herein, that comprises a region or regions that are complementary to the target nucleic acid. In another embodiment, a substrate of a flap-specific nuclease according to the invention comprises a target nucleic acid, an upstream oligonucleotide that is complementary to the target nucleic acid sequence and a downstream probe, according to the invention, that comprises a region or regions that are complementary to the target nucleic acid. In one embodiment, the upstream oligonucleotide and the downstream probe hybridize to non-overlapping regions of the target nucleic acid. In another embodiment the upstream oligonucleotide and the downstream probe hybridize to adjacent regions of the target nucleic acid.
As used herein, xe2x80x9cadjacentxe2x80x9d refers to separated by less than 20 nucleotides, e.g., 15 nucleotides, 10 nucleotides, 5 nucleotides or 0 nucleotides.
A substrate of a flap-specific nuclease according to the invention, also comprises a target nucleic acid, a second nucleic acid, a portion of which specifically hybridizes with a target nucleic acid, and a primer extension product from a third nucleic acid that specifically hybridizes with a target nucleic acid sequence.
As used herein, a xe2x80x9ccleavage structurexe2x80x9d refers to a polynucleotide structure (for example as illustrated in FIG. 1) comprising at least a duplex nucleic acid having a single stranded region comprising a flap, a loop, a single-stranded bubble, a D-loop, a nick or a gap. A cleavage structure according to the invention thus includes a polynucleotide structure comprising a flap strand of a branched DNA wherein a 5xe2x80x2 single-stranded polynucleotide flap extends from a position near its junction to the double stranded portion of the structure and preferably the flap is labeled with a detectable label. A flap of a cleavage structure according to the invention is preferably about 1-500 nucleotides, more preferably about 5-25 nucleotides and most preferably about 10-20 nucleotides and is preferably cleaved at a position located either one nucleotide proximal and/or one nucleotide distal from the elbow of the flap strand. In one embodiment, a flap of a cleavage structure cannot hybridize to a target nucleic acid sequence.
A cleavage structure according to one embodiment of the invention preferably comprises a target nucleic acid sequence, and also may include an oligonucleotide probe according to the invention, that specifically hybridizes with the target nucleic acid sequence via a region or regions that are complementary to the target nucleic acid, and a flap extending from the hybridizing oligonucleotide probe. In another embodiment of the invention, a cleavage structure comprises a target nucleic acid sequence (for example B in FIG. 4), an upstream oligonucleotide that is complementary to the target sequence (for example A in FIG. 4), and a downstream oligonucleotide probe according to the invention and comprisinga region or regions, that are complementary to the target sequence (for example C in FIG. 4). In one embodiment, the upstream oligonucleotide and the downstream probe hybridize to non-overlapping regions of the target nucleic acid. In another embodiment, the upstream oligonucleotide and the downstream probe hybridize to adjacent regions of the target nucleic acid.
A cleavage structure according to the invention may be a polynucleotide structure comprising a flap extending from the downstream oligonucleotide probe of the invention, wherein the flap is formed by extension of the upstream oligonucleotide by the synthetic activity of a nucleic acid polymerase, and subsequent, partial, displacement of the 5xe2x80x2 end of the downstream oligonucleotide. In such a cleavage structure, the downstream oligonucleotide may be blocked at the 3xe2x80x2 terminus to prevent extension of the 3xe2x80x2 end of the downstream oligonucleotide.
A cleavage structure according to one embodiment of the invention may be formed by hybridizing a target nucleic acid sequence with an oligonucleotide probe wherein the oligonucleotide probe has a secondary structure that changes upon binding of the probe to the target nucleic acid, and further comprises a complementary region that anneals to the target nucleic acid sequence, and a non-complementary region that does not anneal to the target nucleic acid sequence and forms a 5xe2x80x2 flap.
A cleavage structure also may be a pseudo-Y structure wherein a pseudo Y-structure is formed if the strand upstream of a flap (referred to herein as a flap adjacent strand or primer strand) is not present, and double stranded DNA substrates containing a gap or nick. A xe2x80x9ccleavage structurexe2x80x9d, as used herein, does not include a double stranded nucleic acid structure with only a 3xe2x80x2 single-stranded flap. As used herein, a xe2x80x9ccleavage structurexe2x80x9d comprises ribonucleotides or deoxyribonucleotides and thus can be RNA or DNA.
A cleavage structure according to the invention may be an overlapping flap wherein the 3xe2x80x2 end of an upstream oligonucleotide capable of hybridizing to a target nucleic acid sequence (for example A in FIG. 4) is complementary to 1 base pair of the downstream oligonucleotide probe of the invention (for example C in FIG. 4) that is annealed to a target nucleic acid sequence and wherein the overlap is directly downstream of the point of extension of the single stranded flap.
A cleavage structure according to the invention is formed by the steps of 1. incubating a) an upstream oligonucleotide, b) an oligonucleotide probe located not more than 5000 nucleotides downstream of the upstream primer and having a secondary structure that changes upon binding of the probe to the target nucleic acid c) an appropriate target nucleic acid sequence wherein the target sequence is complementary to both the upstream primer and downstream probe and d) a suitable buffer, under conditions that allow the nucleic acid sequence to hybridize to the oligonucleotide primers, and, in one embodiment of the invention, 2. extending the 3xe2x80x2 end of the upstream oligonucleotide primer by the synthetic activity of a polymerase such that the newly synthesized 3xe2x80x2 end of the upstream oligonucleotide primer becomes adjacent to and/or displaces at least a portion of (i.e., at least 1-10 nucleotides of) the 5xe2x80x2 end of the downstream oligonucleotide probe. According to the method of the invention, buffers and extension temperatures are favorable for strand displacement by a particular nucleic acid polymerase according to the invention. Preferably, the downstream oligonucleotide is blocked at the 3xe2x80x2 terminus to prevent extension of the 3xe2x80x2 end of the downstream oligonucleotide.
In another embodiment of the invention, a cleavage structure according to the invention can be prepared by incubating a target nucleic acid sequence with an oligonucleotide probe having a secondary structure that changes upon binding of the probe to the target nucleic acid, and further comprising a non-complementary 5xe2x80x2 region that does not anneal to the target nucleic acid sequence and forms a 5xe2x80x2 flap, and a complementary 3xe2x80x2 region that anneals to the target nucleic acid sequence.
In another embodiment of the invention, a cleavage structure according to the invention can be prepared by incubating a target nucleic acid sequence with a downstream oligonucleotide probe having a secondary structure that changes upon binding of the probe to the target nucleic acid, and further comprising a non-complementary 5xe2x80x2 region that does not anneal to the target nucleic acid sequence and forms a 5xe2x80x2 flap and a complementary 3xe2x80x2 region that anneals to the target nucleic acid sequence, and an upstream oligonucleotide. In one embodiment, the upstream oligonucleotide and the downstream probe hybridize to non-overlapping regions of the target nucleic acid. In another embodiment, the upstream oligonucleotide and the downstream probe hybridize to adjacent regions of the target nucleic acid.
In a preferred embodiment of the invention a cleavage structure is labeled. A labeled cleavage structure according to one embodiment of the invention is formed by the steps of 1. incubating a) an upstream extendable 3xe2x80x2 end, for example, an oligonucleotide primer, b) a labeled probe having a secondary structure that changes upon binding of the probe to the target nucleic acid, preferably located not more than 5000 and more preferably located not more than 500 nucleotides downstream of the upstream primer and c) an appropriate target nucleic acid sequence wherein the target sequence is complementary to both the primer and the labeled probe and d) a suitable buffer, under conditions that allow the nucleic acid sequence to hybridize to the primers, and, in one embodiment of the invention, 2. extending the 3xe2x80x2 end of the upstream primer by the synthetic activity of a polymerase such that the newly synthesized 3xe2x80x2 end of the upstream primer partially displaces the 5xe2x80x2 end of the downstream probe. According to the method of the invention, buffers and extension temperatures are favorable for strand displacement by a particular nucleic acid polymerase according to the invention. Preferably, the downstream oligonucleotide is blocked at the 3xe2x80x2 terminus to prevent extension of the 3xe2x80x2 end of the downstream oligonucleotide. In one embodiment, the upstream primer and the downstream probe hybridize to non-overlapping regions of the target nucleic acid.
In another embodiment, a cleavage structure according to the invention can be prepared by incubating a target nucleic acid sequence with a probe having a secondary structure that changes upon binding of the probe to the target nucleic acid, and further comprising a non-complementary, labeled, 5xe2x80x2 region that does not anneal to the target nucleic acid sequence and forms a 5xe2x80x2 flap, and a complementary 3xe2x80x2 region that anneals to the target nucleic acid sequence.
In another embodiment, a cleavage structure according to the invention can be prepared by incubating a target nucleic acid sequence with a downstream probe having a secondary structure that changes upon binding of the probe to the target nucleic acid, and further comprising a non-complementary, labeled, 5xe2x80x2 region that does not anneal to the target nucleic acid sequence and forms a 5xe2x80x2 flap and a complementary 3xe2x80x2 region that anneals to the target nucleic acid sequence, and an upstream oligonucleotide primer. In one embodiment, the upstream oligonucleotide and the downstream probe hybridize to non-overlapping regions of the target nucleic acid. In another embodiment, the upstream oligonucleotide and the downstream probe hybridize to adjacent regions of the target nucleic acid.
As used herein, xe2x80x9clabelxe2x80x9d or xe2x80x9clabeled moiety capable of providing a signalxe2x80x9d refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be operatively linked to a nucleic acid. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, mass spectrometry, binding affinity, hybridization radiofrequency, nanocrystals and the like. A labeled probe according to the methods of the invention is labeled at the 5xe2x80x2 end, the 3xe2x80x2 end or internally. The label can be xe2x80x9cdirectxe2x80x9d, i.e. a dye, or xe2x80x9cindirectxe2x80x9d. i.e. biotin, digoxin, alkaline phosphatase (AP), horse radish peroxidase (HRP) etc . . . For detection of xe2x80x9cindirect labelsxe2x80x9d it is necessary to add additional components such as labeled antibodies, or enzyme substrates to visualize the, released, labeled nucleic acid fragment.
In one embodiment of the invention, a label cannot provide a detectable signal unless the secondary structure has xe2x80x9cchangedxe2x80x9d, as defined herein (for example, such that the label is accessible).
As used herein, xe2x80x9cgenerating a signalxe2x80x9d refers to detecting and or measuring a released nucleic acid fragment that is released from the cleavage structure, as an indication of the presence of a target nucleic acid sequence in a sample.
As used herein, xe2x80x9csamplexe2x80x9d refers to any substance containing or presumed to contain a nucleic acid of interest (a target nucleic acid sequence) or which is itself a nucleic acid containing or presumed to contain a target nucleic acid sequence of interest. The term xe2x80x9csamplexe2x80x9d thus includes a sample of nucleic acid (genomic DNA, cDNA, RNA), cell, organism, tissue, fluid, or substance including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, stool, external secretions of the skin, respiratory, intestinal and genitourinary tracts, saliva, blood cells, tumors, organs, tissue, samples of in vitro cell culture constituents, natural isolates (such as drinking water, seawater, solid materials), microbial specimens, and objects or specimens that have been xe2x80x9cmarkedxe2x80x9d with nucleic acid tracer molecules.
As used herein, xe2x80x9ctarget nucleic acid sequencexe2x80x9d or xe2x80x9ctemplate nucleic acid sequencexe2x80x9d refers to a region of a nucleic acid that is to be either replicated, amplified, and/or detected. In one embodiment, the xe2x80x9ctarget nucleic acid sequencexe2x80x9d or xe2x80x9ctemplate nucleic acid sequencexe2x80x9d resides between two primer sequences used for amplification.
As used herein, xe2x80x9cnucleic acid polymerasexe2x80x9d refers to an enzyme that catalyzes the polymerization of nucleoside triphosphates. Generally, the enzyme will initiate synthesis at the 3xe2x80x2-end of the primer annealed to the target sequence, and will proceed in the 5xe2x80x2-direction along the template, and if possessing a 5xe2x80x2 to 3xe2x80x2 nuclease activity, hydrolyzing intervening, annealed probe to release both labeled and unlabeled probe fragments, until synthesis terminates. Known DNA polymerases include, for example, E. coli DNA polymerase I, T7 DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Bacillus stearothermophilus DNA polymerase, Thermococcus litoralis DNA polymerase, Thermus aquaticus (Taq) DNA polymerase and Pyrococcus furiosus (Pfu) DNA polymerase.
As used herein, xe2x80x9c5xe2x80x2 to 3xe2x80x2 exonuclease activityxe2x80x9d or xe2x80x9c5xe2x80x2xe2x86x923xe2x80x2 exonuclease activityxe2x80x9d refers to that activity of a template-specific nucleic acid polymerase e.g. a 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity traditionally associated with some DNA polymerases whereby mononucleotides or oligonucleotides are removed from the 5xe2x80x2 end of a polynucleotide in a sequential manner, (i.e., E. coli DNA polymerase I has this activity whereas the Klenow (Klenow et al., 1970, Proc. Natl. Acad. Sci., USA, 65:168) fragment does not, (Klenow et al., 1971, Eur. J. Biochem., 22:371)), or polynucleotides are removed from the 5xe2x80x2 end by an endonucleolytic activity that may be inherently present in a 5xe2x80x2 to 3xe2x80x2 exonuclease activity.
As used herein, the phrase xe2x80x9csubstantially lacks 5xe2x80x2 to 3xe2x80x2 exonuclease activityxe2x80x9d or xe2x80x9csubstantially lacks 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activityxe2x80x9d means having less than 10%, 5%, 1%, 0.5%, or 0.1% of the activity of a wild type enzyme. The phrase xe2x80x9clacking 5xe2x80x2 to 3xe2x80x2 exonuclease activityxe2x80x9d or xe2x80x9clacking 5xe2x86x92xe2x80x23xe2x80x2 exonuclease activityxe2x80x9d means having undetectable 5xe2x80x2 to 3xe2x80x2 exonuclease activity or having less than about 1%, 0.5%, or 0.1% of the 5xe2x80x2 to 3xe2x80x2 exonuclease activity of a wild type enzyme. 5xe2x80x2 to 3xe2x80x2 exonuclease activity may be measured by an exonuclease assay which includes the steps of cleaving a nicked substrate in the presence of an appropriate buffer, for example 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2 and 50 xcexcg/ml bovine serum albumin) for 30 minutes at 60xc2x0 C., terminating the cleavage reaction by the addition of 95% formamide containing 10 mM EDTA and 1 mg/ml bromophenol blue, and detecting nicked or un-nicked product. Nucleic acid polymerases useful according to the invention include but are not limited to any enzyme that possesses 5xe2x80x2 endonucleolytic activity for example a DNA polymerase, e.g. DNA polymerase I from E. coli, and DNA polymerase from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Pyrococcus furiosus (Pfu) and Thermus flavus (Tfl), Pfu, exo- Pfu (a mutant form of Pfu that lacks 3xe2x80x2 to 5xe2x80x2 exonuclease activity), the Stoffel fragment of Taq, N-truncated Bst, N-truncated Bca, Genta, JdF3 exo-, Vent, Vent exo- (a mutant form of Vent that lacks 3xe2x80x2 to 5xe2x80x2 exonuclease activity), Deep Vent, Deep Vent exo- (a mutant form of Deep Vent that lacks 3xe2x80x2 to 5xe2x80x2 exonuclease activity), U1Tma and Sequenase. Additional nucleic acid polymerases useful according to the invention are included below in the section entitled, xe2x80x9cNucleic Acid Polymerasesxe2x80x9d.
As used herein, xe2x80x9ccleavingxe2x80x9d refers to enzymatically separating a cleavage structure into distinct (i.e. not physically linked to other fragments or nucleic acids by phosphodiester bonds) fragments or nucleotides and fragments that are released from the cleavage structure. For example, cleaving a labeled cleavage structure refers to separating a labeled cleavage structure according to the invention and defined below, into distinct fragments including fragments derived from an oligonucleotide that specifically hybridizes with a target nucleic acid sequence or wherein one of the distinct fragments is a labeled nucleic acid fragment derived from a target nucleic acid sequence and/or derived from an oligonucleotide that specifically hybridizes with a target nucleic acid sequence that can be detected and/or measured by methods well known in the art and described herein that are suitable for detecting the labeled moiety that is present on a labeled fragment.
As used herein, xe2x80x9cendonucleasexe2x80x9d refers to an enzyme that cleaves bonds, preferably phosphodiester bonds, within a nucleic acid molecule. An endonuclease according to the invention can be specific for single-stranded or double-stranded DNA or RNA.
As used herein, xe2x80x9cexonucleasexe2x80x9d refers to an enzyme that cleaves bonds, preferably phosphodiester bonds, between nucleotides one at a time from the end of a polynucleotide. An exonuclease according to the invention can be specific for the 5xe2x80x2 or 3xe2x80x2 end of a DNA or RNA molecule, and is referred to herein as a 5xe2x80x2 exonuclease or a 3xe2x80x2 exonuclease.
As used herein a xe2x80x9cflapxe2x80x9d refers to a region of single stranded DNA that extends from a double stranded nucleic acid molecule. A flap according to the invention is preferably between about 1-500 nucleotides, more preferably between about 5-25 nucleotides and most preferably between about 10-20 nucleotides.
In a preferred embodiment, the signal is detected or measured, wherein detecting and/or measuring the signal comprises detecting and/or measuring the amount of the fragment.
The invention also provides a method of detecting or measuring a target nucleic acid sequence comprising the steps of: forming a cleavage structure by incubating a sample containing a target nucleic acid sequence with a probe having a secondary structure that changes upon binding of the probe to the target nucleic acid sequence, cleaving the cleavage structure with a nuclease to release a nucleic acid fragment wherein the cleavage is performed at a cleaving temperature, and the secondary structure of the probe when not bound to the target nucleic acid sequence is stable at or below the cleaving temperature; and detecting and/or measuring the amount of the fragment as an indication of the presence of the target sequence in the sample.
As used herein, xe2x80x9cdetecting a target nucleic acid sequencexe2x80x9d or xe2x80x9cmeasuring a target nucleic acid sequencexe2x80x9d refers to determining the presence of a particular target nucleic acid sequence in a sample or determining the amount of a particular target nucleic acid sequence in a sample as an indication of the presence of a target nucleic acid sequence in a sample. The amount of a target nucleic acid sequence that can be measured or detected is preferably about 1 molecule to 1020 molecules, more preferably about 100 molecules to 1017 molecules and most preferably about 1000 molecules to 1014 molecules. According to one embodiment of the invention, the detected nucleic acid is derived from the labeled 5xe2x80x2 end of a downstream probe of a cleavage structure according to the invention (for example C in FIG. 4), that is displaced from the target nucleic acid sequence by the 3xe2x80x2 extension of an upstream probe of a cleavage structure according to the invention (for example A of FIG. 4). According to the present invention, a label is attached to the 5xe2x80x2 end of the downstream probe (for example C in FIG. 4) comprising a cleavage structure according to the invention. Alternatively, a label is attached to the 3xe2x80x2 end of the downstream probe and a quencher is attached to the 5xe2x80x2 flap of the downstream probe. According to the invention, a label may be attached to the 3xe2x80x2 end of the downstream probe (for example C in FIG. 4) comprising a cleavage structure according to the invention.
According to the invention, the downstream probe (for example C in FIG. 4) may be labeled internally. In a preferred embodiment, a cleavage structure according to the invention can be prepared by incubating a target nucleic acid sequence with a probe having a secondary structure that changes upon binding of the probe to the target nucleic acid sequence, and further comprising a non-complementary, labeled, 5xe2x80x2 region that does not anneal to the target nucleic acid sequence and forms a 5xe2x80x2 flap, and a complementary 3xe2x80x2 region that anneals to the target nucleic acid sequence. According to this embodiment of the invention, the detected nucleic acid is derived from the labeled 5xe2x80x2 flap region of the probe. Preferably there is a direct correlation between the amount of the target nucleic acid sequence and the signal generated by the cleaved, detected nucleic acid.
In another embodiment of the invention, the probe is labeled with a pair of interactive labels (e.g., a FRET or non-FRET pair) positioned to permit the separation of the labels during oligonucleotide probe unfolding (e.g., for example due to a change in the secondary structure of the probe) or hydrolysis.
As used herein, xe2x80x9cdetecting the amount of the fragmentxe2x80x9d or xe2x80x9cmeasuring the amount of the fragmentxe2x80x9d refers to determining the presence of a labeled fragment in a sample or determining the amount of a labeled fragment in a sample. Methods well known in the art and described herein can be used to detect or measure release of labeled fragments. A method of detecting or measuring release of labeled fragments will be appropriate for measuring or detecting the labeled moiety that is present on the labeled fragments. The amount of a released labeled fragment that can be measured or detected is preferably about 25%, more preferably about 50% and most preferably about 95% of the total starting amount of labeled probe.
As used herein, xe2x80x9clabeled fragmentsxe2x80x9d refer to cleaved mononucleotides or small oligonucleotides or oligonucleotides derived from the labeled cleavage structure according to the invention wherein the cleaved oligonucleotides are preferably between about 2-1000 nucleotides, more preferably between about 5-50 nucleotides and most preferably between about 16-18 nucleotides, which are cleaved from a cleavage structure by a nuclease and can be detected by methods well known in the art and described herein.
In one embodiment, a probe is a bi-molecular or multimolecular probe wherein a first molecule comprising the probe is labeled with a fluorophore and a second molecule comprising the probe is labeled with a quencher. As used herein, a xe2x80x9csubprobexe2x80x9d and xe2x80x9csubquencherxe2x80x9d refer to a first molecule of a bi- or multi-molecular probe according to the invention, that is labeled with a fluorophore and a second molecule of a bi- or multi-molecular probe according to the invention, that is labeled with a quencher, respectively. According to this embodiment, following binding of the bi- or multi-molecular probe to the target nucleic acid, and cleavage by a nuclease, the subprobe and subquencher dissociate from each other (that is, the distance between the subprobe and the subquencher increases) and a signal is generated as a result of this dissociation and subsequent separation of the subprobe and subquencher.
In a preferred embodiment, the method further comprises a nucleic acid polymerase.
In a preferred embodiment, the cleavage structure further comprises an oligonucleotide primer.
In another preferred embodiment, the cleavage structure further comprises a 5xe2x80x2 flap.
In another preferred embodiment, the cleavage structure further comprises an oligonucleotide primer.
In another preferred embodiment, the secondary structure is selected from the group consisting a stem-loop structure, a hairpin structure, an internal loop, a bulge loop, a branched structure, a pseudoknot structure or a cloverleaf structure.
In another preferred embodiment, the nuclease is a FEN nuclease.
In another preferred embodiment the FEN nuclease is selected from the group consisting of FEN nuclease enzyme derived from Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus, human, mouse or Xenopus laevis. A FEN nuclease according to the invention also includes Saccharomyces cerevisiae RAD27, and Schizosaccharomyces pombe RAD2, Pol I DNA polymerase associated 5xe2x80x2 to 3xe2x80x2 exonuclease domain, (e.g. E. coli, Thermus aquaticus (Taq), Thermus flavus (Tfl), Bacillus caldotenax (Bca), Streptococcus pneumoniae) and phage functional homologs of FEN including but not limited to T4, T5 5xe2x80x2 to 3xe2x80x2 exonuclease, T7 gene 6 exonuclease and T3 gene 6 exonuclease.
Preferably, only the 5xe2x80x2 to 3xe2x80x2 exonuclease domains of Taq, Tfl and Bca FEN nuclease are used.
In another preferred embodiment, the cleavage structure is formed comprising at least one labeled moiety capable of providing a signal.
In another preferred embodiment, the cleavage structure is formed comprising a pair of interactive signal generating labeled moieties effectively positioned on the probe to quench the generation of a detectable signal when the probe is not bound to the target nucleic acid.
In another preferred embodiment, the labeled moieties are separated by a site susceptible to nuclease cleavage, thereby allowing the nuclease activity of the nuclease to separate the first interactive signal generating labeled moiety from the second interactive signal generating labeled moiety by cleaving at the site susceptible to nuclease cleavage, thereby generating a detectable signal.
In another preferred embodiment, the pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.
The invention also provides for a polymerase chain reaction process for detecting a target nucleic acid sequence in a sample comprising; providing a cleavage structure comprising a probe having a secondary structure that changes upon binding of the probe to the target nucleic acid sequence, a set of oligonucleotide primers wherein a first primer contains a sequence complementary to a region in one strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and a second primer contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand; and amplifying the target nucleic acid sequence employing a nucleic acid polymerase as a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers required for amplification to a template nucleic acid sequence contained within the target nucleic acid sequence, (ii) extending the primers wherein the nucleic acid polymerase synthesizes a primer extension product, and (iii) cleaving the cleavage structure employing a nuclease as a cleavage agent for release of labeled fragments from the cleavage structure thereby creating detectable labeled fragments; wherein the cleaving is performed at a cleaving temperature and the secondary structure of the second primer when not bound to the target nucleic acid sequence is stable at or below the cleaving temperature; and detecting and/or measuring the amount of released, labeled fragment.
As used herein, an xe2x80x9coligonucleotide primerxe2x80x9d refers to a single stranded DNA or RNA molecule that is hybridizable to a nucleic acid template and primes enzymatic synthesis of a second nucleic acid strand. Oligonucleotide primers useful according to the invention are between about 10 to 100 nucleotides in length, preferably about 17-50 nucleotides in length and more preferably about 17-45 nucleotides in length. Oligonucleotide probes useful for the formation of a cleavage structure according to the invention are between about 17-40 nucleotides in length, preferably about 17-30 nucleotides in length and more preferably about 17-25 nucleotides in length.
As used herein, xe2x80x9ctemplate dependent polymerizing agentxe2x80x9d refers to an enzyme capable of extending an oligonucleotide primer in the presence of adequate amounts of the four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP and dTTP) or analogs as described herein, in a reaction medium comprising appropriate salts, metal cations, appropriate stabilizers and a pH buffering system. Template dependent polymerizing agents are enzymes known to catalyze primer- and template-dependent DNA synthesis, and possess 5xe2x80x2 to 3xe2x80x2 nuclease activity. Preferably, a template dependent polymerizing agent according to the invention lacks 5xe2x80x2 to 3xe2x80x2 nuclease activity.
As used herein, xe2x80x9camplifyingxe2x80x9d refers to producing additional copies of a nucleic acid sequence, including the method of the polymerase chain reaction.
In a preferred embodiment, the nuclease is a FEN nuclease.
In another preferred embodiment, the oligonucleotide primers of step b are oriented such that the forward primer is located upstream of the cleavage structure and the reverse primer is located downstream of the cleavage structure.
In another preferred embodiment, the nucleic acid polymerase has strand displacement activity. Nucleic acid polymerases exhibiting strand displacement activity and useful according to the invention include but are not limited to 9xc2x0N DNA polymerase, Vent DNA polymerase, Vent(exo-) DNA polymerase, the Klenow fragment of DNA polymerase I, and the 5xe2x80x2 to 3xe2x80x2 exo- Klenow fragment of DNA polymerase I. In another preferred embodiment, the nucleic acid polymerase is thermostable.
In another preferred embodiment, the nuclease is thermostable.
As used herein, xe2x80x9cthermostablexe2x80x9d refers to an enzyme which is stable and active at temperatures as great as preferably between about 90-100xc2x0 C. and more preferably between about 70-98xc2x0 C. to heat as compared, for example, to a non-thermostable form of an enzyme with a similar activity. For example, a thermostable nucleic acid polymerase or FEN nuclease derived from thermophilic organisms such as P. furiosus, M. jannaschii, A. fulgidus or P. horikoshii are more stable and active at elevated temperatures as compared to a nucleic acid polymerase from E. coli or a mammalian FEN enzyme. A representative thermostable nucleic acid polymerase isolated from Thermus aquaticus (Taq) is described in U.S. Pat. No. 4,889,818 and a method for using it in conventional PCR is described in Saiki et al., 1988, Science 239:487. Another representative thermostable nucleic acid polymerase isolated from P. furiosus (Pfu) is described in Lundberg et al., 1991, Gene, 108:1-6. Additional representative temperature stable polymerases include, e.g., polymerases extracted from the thermophilic bacteria Thermus flavus, Thermus ruber, Thermus thermophilus, Bacillus stearothermophilus (which has a somewhat lower temperature optimum than the others listed), Thermus lacteus, Thermus rubens, Thermotoga maritima, or from thermophilic archaea Thermococcus litoralis, and Methanothermus fervidus.
Temperature stable polymerases and FEN nucleases are preferred in a thermocycling process wherein double stranded nucleic acids are denatured by exposure to a high temperature (about 95xc2x0 C.) during the PCR cycle.
In another preferred embodiment, the nuclease is a flap-specific nuclease.
In another preferred embodiment, the cleavage structure is formed comprising at least one labeled moiety capable of providing a signal.
In another preferred embodiment, the cleavage structure is formed comprising a pair of interactive signal generating labeled moieties effectively positioned on the probe to quench the generation of a detectable signal when the probe is not bound to the target nucleic acid.
In another preferred embodiment, the labeled moieties are separated by a site susceptible to nuclease cleavage, thereby allowing the nuclease activity of the nuclease to separate the first interactive signal generating labeled moiety from the second interactive signal generating labeled moiety by cleaving at the site susceptible to nuclease cleavage, thereby generating a detectable signal.
In another preferred embodiment, the pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.
In another preferred embodiment, the nucleic acid polymerase is selected from the group consisting of Taq polymerase and Pfu polymerase.
The invention provides for a polymerase chain reaction process wherein amplification and detection of a target nucleic acid sequence occur concurrently (i.e., real time detection). The invention also provides for a polymerase chain reaction process wherein amplification of a target nucleic acid sequence occurs prior to detection of the target nucleic acid sequence (i.e., end point detection).
The invention also provides for a polymerase chain reaction process for simultaneously forming a cleavage structure, amplifying a target nucleic acid sequence in a sample and cleaving the cleavage structure comprising the steps of: (a) providing an upstream oligonucleotide primer complementary to a first region in one strand of the target nucleic acid sequence, a downstream labeled probe complementary to a second region in the same strand of the target nucleic acid sequence, wherein the downstream labeled probe is capable of forming a secondary structure that changes upon binding of the probe to the target nucleic acid sequence, and a downstream oligonucleotide primer complementary to a region in a second strand of the target nucleic acid; and wherein the upstream primer primes the synthesis of a complementary DNA strand, and the downstream primer primes the synthesis of a complementary DNA strand; and (b) detecting a nucleic acid which is produced in a reaction comprising amplification of the target nucleic acid sequence and cleavage thereof wherein a nucleic acid polymerase is a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers to a target nucleic acid sequence, (ii) extending the primers of step (a), wherein the nucleic acid polymerase synthesizes primer extension products, and wherein the primer extension product of the upstream primer of step (a) partially displaces the downstream probe of step (a) to form a cleavage structure; and (iii) cleaving the cleavage structure employing a nuclease as a cleavage agent for release of labeled fragments from the cleavage structure, wherein the cleaving is performed at a cleaving temperature and the secondary structure of the probe when not bound to the target nucleic acid sequence is stable at or below the cleaving temperature, thereby creating detectable labeled fragments.
In a preferred embodiment, the cleavage structure further comprises a 5xe2x80x2 flap.
The invention also provides a method of forming a cleavage structure comprising the steps of: (a) providing a target nucleic acid sequence, (b) providing an upstream primer complementary to the target nucleic acid sequence, (c) providing a downstream probe having a secondary structure that changes upon binding of the probe to a target nucleic acid sequence; and (d) annealing the target nucleic acid sequence, the upstream primer and the downstream probe and; wherein the cleavage structure can be cleaved with a nuclease at a cleaving temperature, and wherein the secondary structure of the probe when not bound to the target nucleic acid sequence is stable at or below the cleaving temperature.
In a preferred embodiment, the cleavage structure comprises a 5xe2x80x2 flap. The invention also provides for a composition comprising a target nucleic acid sequence, a probe having a secondary structure that changes upon binding of the probe to a target nucleic acid sequence, and a nuclease; and wherein the probe and the target nucleic acid can bind to form a cleavage structure that can be cleaved by the nuclease wherein the cleaving is performed at a cleaving temperature, and wherein the secondary structure of the probe when not bound to the target nucleic acid sequence is stable at or below the cleaving temperature.
In a preferred embodiment, the composition further comprises an oligonucleotide primer.
In another preferred embodiment, the probe and the oligonucleotide hybridize to non-overlapping regions of the target nucleic acid sequence.
The invention also provides for a kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising a probe having a secondary structure that changes upon binding of the probe to a target nucleic acid sequence, and a nuclease; and wherein the probe can bind to a target nucleic acid sequence to form a cleavage structure that can be cleaved by the nuclease; and wherein the cleaving is performed at a cleaving temperature, and wherein the secondary structure of the probe when not bound to the target nucleic acid sequence is stable at or below the cleaving temperature.
In a preferred embodiment, the kit further comprises an oligonucleotide primer. In another preferred embodiment, the nuclease is a FEN nuclease.
In another preferred embodiment, the probe comprises at least one labeled moiety.
In another preferred embodiment, the probe comprises a pair of interactive signal generating labeled moieties effectively positioned to quench the generation of a detectable signal when the probe is not bound to the target nucleic acid.
In another preferred embodiment, the labeled moieties are separated by a site susceptible to nuclease cleavage, thereby allowing the nuclease activity of the nuclease to separate the first interactive signal generating labeled moiety from the second interactive signal generating labeled moiety by cleaving at the site susceptible to nuclease cleavage, thereby generating a detectable signal.
In another preferred embodiment, the pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.
Further features and advantages of the invention are as follows. The claimed invention provides a method of generating a signal to detect and/or measure a target nucleic acid wherein the generation of a signal is an indication of the presence of a target nucleic acid in a sample. The method of the claimed invention does not require multiple steps. The claimed invention also provides a PCR based method for detecting and/or measuring a target nucleic acid comprising generating a signal as an indication of the presence of a target nucleic acid. The claimed invention allows for simultaneous amplification and detection and/or measurement of a target nucleic acid sequence. The claimed invention also provides a PCR based method for detecting and/or measuring a target nucleic acid comprising generating a signal in the absence of a nucleic acid polymerase that demonstrates 5xe2x80x2 to 3xe2x80x2 exonuclease activity.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.

Methods for detection of a target nucleic acid using a probe comprising secondary structure


A printed wiring board includes a first insulating layer, a second conductor layer including first and second circuits, a second insulating layer covering the second conductor layer on the first insulating layer, a third conductor layer including first and second circuits, a third insulating layer covering the third conductor layer on the second insulating layer, a fourth conductor layer including first circuit, a second via conductor connecting the first circuits in the second and third conductor layers through the second insulating layer, and a first skip via conductor penetrating through the second circuit in the third conductor layer and connecting the second circuit in the second conductor layer and the first circuit in the fourth conductor layer through the second and third insulating layers. The second and third conductor layers are formed such that the second conductor layer has thickness t2 larger than thickness t3 of the third conductor layer.

Field of the Invention
The present invention relates to a printed wiring board having a skip via conductor that penetrates multiple resin insulating layers.
Description of Background Art
Japanese Patent Laid-Open Publication No. 2007-115954 describes a multilayer printed wiring board that has a via that penetrates one insulating layer and a skip via that penetrates multiple insulating layers. The entire contents of this publication are incorporated herein by reference.


A comprehensive authentication and identity system and method are disclosed. A central profile is created for a user which includes user information that can be passed back or otherwise utilized by websites (e.g. for registrations, logins, etc.) The user information may include the user's username, password, contact information, personal information, marketing preferences, financial information, etc. For website registrations, the user may provide a mobile communication number that is utilized to perform a type of mobile communication device verification process. As part of a website login, the user may provide identifiable information (e.g. a username) that is looked up by the system or website to determine a mobile communication number for the user, which is used for a verification process. If the verification process is completed successfully, the user may be logged into the website. For accessing the system directly, a user may go through a mobile communication device verification process.

One challenge for individuals transacting on the World Wide Web is that separate credentials are typically needed (typically a unique user name and password) for each website that is visited. The use of separate credentials requires individuals to store a variety of usernames and passwords. All too often people write down passwords on a piece of paper or store them in a word processing document on their computer, which makes the passwords vulnerable to identity thieves. Several companies have attempted to develop solutions to this problem. For example, a company named LastPass allows a consumer to store all of his or her passwords in one place and then allows for an expedited login process using the LastPass application. One drawback with LastPass's default settings is that if someone breaches the consumer's local computer and LastPass account, they will be able to break into all of the accounts that are stored within the LastPass user's encrypted vault. A similar problem arises when an individual uses the same username and password across various websites. If one website is breached by a hacker, the hacker may be able to use the stolen credentials across a variety of other sites. As an example of the potential severity of what might occur, Zappos.com had as many as 24 million usernames and password combinations breached in early 2012 which could have led to many other websites being accessed under stolen username/password combinations. And more recently, attacks based on social engineering can be used to reset account passwords where only public information is leveraged to obtain account access. This type of attack led to one Apple user's entire group of devices being remotely wiped of all of his information.
Another problem with current solutions for registering with websites is the challenge that a user faces to quickly and easily complete the registration process. While tools like "auto fill" in website browsers alleviate some registration issues, they don't help if a computer is shared with other users or if some of the data that needs to be entered for registration purposes is confidential and/or sensitive. For example, in most cases a user would not want an auto filler tool storing their credit card number.
The problems associated with managing login credentials have been recognized by a U.S. government program called "The National Strategy for Trusted Identities in Cyberspace (NSTIC)" which strives to "improve upon the passwords currently used to log-in online." The NSTIC report highlights that "a contributing factor is the unmanageable number of passwords people must remember to access their online accounts. Many people don't even try; they just re-use the same ones for all of their accounts, making it that much easier for identity thieves" (www.nist.gov/nstic/index.html). Additionally, prior art systems like OpenID allow a person to use one set of credentials to log onto other websites (e.g., Yahoo or Facebook). While this indicates that a user doesn't have to keep track of as many credentials, it still results in a situation where if someone breaches those credentials then they would have access to multiple accounts belonging to a user. The NSTIC goes on to state that the new identity systems should be "faster, more convenient, safer, private, and voluntary." Accordingly, there is a need for a comprehensive identity system that allows easy and secure registration and access to websites while maintaining a high level of security.

This patent is subject to a terminal disclaimer.

Comprehensive authentication and identity system and method


A method of fabricating a component for a gas turbine engine is provided. The method includes applying a bond coat to at least a portion of the component, applying a dense vertically cracked (DVC) thermal barrier coating to at least a portion of the bond coat using a spray mechanism positioned a first distance from the component, and overlying at least a portion of the DVC thermal barrier coating with a soft coat thermal barrier coating using a spray mechanism that is positioned a second distance away from the component, wherein the second distance is greater than the first distance to facilitate adherence of the soft coating thermal barrier coating to the DVC thermal barrier coating.

This invention relates generally to gas turbine engines, and more particularly to methods and apparatus for fabricating a coating for gas turbine engine blades.
At least some known gas turbine engines include one or more turbine rotors, which include a rotor disk and a plurality of circumferentially-spaced rotor blades, sometimes referred to as blades. Known blades typically include an airfoil, a platform, a shank, and a dovetail. Each dovetail is received within a slot or opening defined in the rotor disk to secure the blades to the rotor disk. The airfoils extend from the disk into the flow of combustion gases within the engine and convert kinetic energy of the gas flow into rotational mechanical energy.
Fouling and surface degradation generally occur when as the result of burning fuels containing alkali metals that combine with sulfur during the combustion process and deposit low melting salts on the surface of the parts. Additional contaminants may come from air ingested and/or water injected for NOx control or power augmentation.
To facilitate protecting the blades during engine operation, at least some known turbine engine blades include a thermal barrier coating (TBC). However, known thermal barrier coatings sometimes have a relatively rough outer surface, and as such, some thermal barrier coatings may be prone to erosion caused by, for example, the adherence of contaminants, such as low melting salts or other contaminants that may be generated during combustion. Erosion of the thermal barrier coating may reduce effectiveness of the thermal barrier coating and/or may exacerbate further erosion, which may lead to even further performance degradation.
To facilitate reducing the effects of the rough outer surface of the TBC, at least some thermal barrier coatings are hand-polished to reduce the surface roughness and increase the erosion resistance of the thermal barrier coating. Hand polishing may increase the time, and/or effort, involved in fabricating turbine blades, and/or may increase the likelihood of human error during fabrication of the turbine blades. As a result, an overall cost of fabricating gas turbine engines including hand-polished blades may be higher than costs associated with fabricating other blades that are not hand-polished.

Methods and apparatus for coating gas turbine engines


Dichroic mirrors employed in an optical seeker system to achieve spectral scrimination. The dichroic mirrors are aligned so as to produce a reticle phase difference between different wavelengths of reflected optical energy. Background optic energy is spectrally balanced so no net guidance is given to the seeker.

1. Field of the Invention
The invention relates to the field of radiation physics. More specifically, the invention relates to the field of infrared radiation. More particularly, the invention relates to the guidance of a missile in the presence of background radiation. In still greater particularity, the invention uses dichroic mirrors in a missile seeker system to balance interfering background or countermeasure radiation so as to produce no net missile guidance.
2. Description of the Prior Art
The reliability and effectiveness of optical seeker systems in missiles is affected by outside infrared radiation sources such as sunlit backgrounds or countermeasure flares. Various methods and devices for alleviating this problem have been attempted. One approach has been to cancel signals from sunlit backgrounds and countermeasure flares by making the modulation efficiency of a reticle dependent upon the spectral nature of the source.
One system for spectral discrimination in seeker systems used spectral band pass filters as alternate spokes of a reticle. Spectral filters, however, only allow reflection of predetermined wavelengths of optical energy to achieve background suppression and spectral balancing. In addition, the fabrication of a reticle utilizing this concept is extremely difficult.
Spectral discrimination remains a viable concept however and a seeker system utilizing it would give greater accuracy to the seeker system.

Spectral discrimination system for an optical seeker

The United States of America as represented by the Secretary of the Navy


An N-channel transistor is provided as a switch between a high potential power line and a low potential power line. A high-pass filter is constituted by a capacitor and a resistor. When a voltage between the high potential power line and the low potential power line is started to oscillate by a switching operation, the high-pass filter causes a high-pass component thereof to pass, thereby turning ON the N-channel transistor to reduce a ringing.

The present invention relates to a semiconductor integrated circuit and more particularly to a semiconductor integrated circuit which is suitable for a circuit for pulse driving a load such as a class-D amplifier.
As is well known, a class-D amplifier serves to turn ON/OFF a load driving output transistor and to intermittently carry out a conduction to a load. In the intermittent conduction to the load, a current flowing to parasitic inductances provided on a power line or a grounding conductor of the class-D amplifier is drastically changed. Therefore, noise is generated on these parasitic inductances, resulting in a ringing which appears in an output signal of the class-D amplifier. Such a ringing causes a deterioration in reproducing quality of the class-D amplifier, and furthermore, causes damage to the load or the class-D amplifier. For this reason, it is desired to reduce the ringing. Patent Document 1 has proposed a technique for decreasing a time gradient of an output signal waveform of an output transistor. By an application of the technique of this type to the class-D amplifier, the time gradient of the output signal waveform is decreased so that a current flowing to an output transistor can be prevented from being changed suddenly. Thus, it is possible to reduce the ringing.
Patent Document 1: Japanese Patent No. 3152204
However, the technique disclosed in the Patent Document 1 serves to decrease the time gradient of the output signal waveform. For this reason, there is a problem in that an operating speed of the class-D amplifier is sacrificed by the application of the technique. This problem is common to a semiconductor integrated circuit required to drive a load at a high speed and demanded to reduce a ringing in an output signal in addition to the class-D amplifier.

Semiconductor integrated circuit for realizing an amplifier having ringing reduction circuitry


A system and method provides Link Aggregation that internalizes the link aggregation to improve internal traffic capacity, in addition to sharing traffic across external links to connecting packet systems. A system for handling data traffic comprises a plurality of network switches, each network switch having an interface to a packet network, the interface arranged so as to provide at least one link aggregation group, an interface to at least one other network switch, and an interface to each of a plurality of optical network service units, a plurality of optical network service units, each optical network service units having an interface to each of the plurality of network switches and an interface to at least one optical network, wherein each network switch is operable to receive data over a link aggregation group from the packet network and transmit the data to at least one optical network service unit either directly or via another network switch based on an optical network to which the data is destined (or other criteria) Also, each optical network service unit can send upstream data to any port of either network switch- via selected internal paths.

1. Field of the Invention
The present invention relates to a system and method providing for link aggregation features of protection and extra bandwidth, while reducing traffic congestion within the system, in a way not visible to an external packet network.
2. Background of the Prior Art
A Passive Optical Network (PON) is a point-to-multipoint, fiber to the premises network architecture in which unpowered optical splitters are used to enable a single optical fiber to serve multiple premises, typically 32. A PON consists of an Optical Line Termination (OLT) typically at the service provider's central office and a number of Optical Network Units (ONUs) near end users. A PON configuration reduces the amount of fiber and central office equipment required compared with point to point architectures. Downstream signals are broadcast to each premises sharing a fiber. Encryption is used to prevent eavesdropping. Upstream signals are combined using a multiple access protocol, typically time division multiple access (TDMA). The OLTs "range" the ONUs in order to provide time slot assignments for upstream communication and to equalize roundtrip transmission delay between the ONU's on a PON and the OLT.
There are a number of standard types of PON that have been implemented. APON (ATM Passive Optical Network) was the first Passive optical network standard. It was used primarily for business applications, and was based on ATM. BPON (Broadband PON) is a standard based on APON. It adds support for WDM, dynamic and higher upstream bandwidth allocation, and survivability. GPON (Gigabit PON) is an evolution of BPON. It supports higher rates, enhanced security, and choice of Layer 2 protocol (ATM, GEM, Ethernet). GPON represents a significant boost in both the total bandwidth and bandwidth efficiency through the use of larger, variable-length packets. A GPON network delivers up to 2,488 megabits per second (Mbit/s) of downstream bandwidth, and 2,488 Mbit/s of upstream bandwidth. GPON Encapsulation Method (GEM) allows very efficient packaging of user traffic, with frame segmentation to allow for higher Quality of Service (QoS) for delay-sensitive traffic such as voice and video communications.
Among the features of GPON is Link Aggregation. Link Aggregation involves aggregating a plurality of links to form a group of links that may be treated as a single link for most purposes. Current implementations of Link Aggregation provide this function only at the external facing ports of a system to a connecting system. In this invention, the link aggregation function not only provides this capability, but internalizes the functionality to provide improved traffic handling throughout the internals of the system.

Link aggregation with internal load balancing