Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

• the present invention generally relates to probes comprising a reporter molecule and a quencher molecule for use in nucleic acid assays.
• the present invention relates to a universal internal positive control that may be used in reverse transcriptase polymerase chain reaction (RT-PCR) based assays.
• RT-PCR reverse transcriptase polymerase chain reaction
• Reporter molecule and quencher molecule pairs have been incorporated onto oligonucleotide probes in order to monitor, detect, or measure biological events associated with the reporter molecule and quencher molecule being separated or brought within a minimum quenching distance of each other.
• probes have been developed where the intensity of the reporter molecule fluorescence increases due to the separation of the reporter molecule from the quencher molecule. Probes have also been developed which lose their fluorescence because the quencher molecule is brought into proximity with the reporter molecule.
• Reporter molecule and quencher molecule pair probes have been used to monitor hybridization assays and nucleic acid amplification reactions, such as polymerase chain reactions (PCR), by monitoring either the appearance or disappearance of the signal generated by the reporter molecule.
• PCR polymerase chain reactions
• TaqMan® based assays use an oligonucleotide probe having a reporter molecule and quencher molecule pair that specifically anneals to a region of a target polynucleotide "downstream", i.e. in the direction of extension of primer binding sites.
• the reporter molecule and quencher molecule are positioned on the probe sufficiently close to each other such that whenever the reporter molecule is excited, the energy of the excited state nonradiatively transfers to the quencher molecule where it either dissipates nonradiatively or is emitted at a different emission frequency than that of the reporter molecule.
• the probe anneals to the template where it is digested by the 5' ⁇ 3' exonuclease activity of the polymerase.
• the reporter molecule is effectively separated from the quencher molecule such that the quencher molecule is no longer close enough to the reporter molecule to quench the fluorescence of the reporter molecule.
• TaqMan® based assays require internal positive control reagents to help distinguish between samples that are identified as negative because the sample lacks the target sequence and samples that are identified as negative because the presence of a PCR inhibitor.
• a TaqMan® Exogenous Internal Positive Control kit is commercially available from Applied Biosystems (Foster City, CA) to distinguish true target negatives from PCR inhibition. The TaqMan® Exogenous Internal Positive Control kit distinguishes two types of negative results. A negative call for the target sequence and a positive call for the internal positive control (IPC) indicates that no target sequence is present and a negative call for the target sequence and a negative call for the IPC suggests PCR inhibition.
• IPC internal positive control
• TaqMan® Exogenous Internal Positive Control kits allow little flexibility as the kits are made with only one fluorescent dye, VICTM (Applied Biosystems, Foster City, CA), which cannot be used on all TaqMan® chemistry based instruments and the primers and probe in the kit are mixed together by the manufacturer and therefore cannot be completely optimized for use with any PCR amplification product assay.
• VICTM Applied Biosystems, Foster City, CA
• the present invention generally relates to a nucleic acid molecule that may be used as an internal positive control in probe-based nucleic acid assays.
• the present invention relates to an isolated nucleic acid molecule comprising the sequence set forth in SEQ ID NO:49.
• the nucleic acid molecule consists essentially of the sequence set forth in SEQ ID NO:49.
• the nucleic acid molecule consists of the sequence set forth in SEQ ID NO:49.
• the present invention provides an isolated nucleic acid molecule comprising at least 80 consecutive bases of SEQ ID NO: 89 or its complement.
• the isolated nucleic acid molecule comprises SEQ ID NO: 89 or its complement.
• the isolated nucleic acid molecule comprises SEQ ID NO:49 or its complement, SEQ ID NO:90.
• the isolated nucleic acid molecule consists essentially of SEQ ID NO: 89 or its complement, hi some embodiments, the isolated nucleic acid molecule consists essentially of SEQ ID NO:49 or its complement, SEQ ID NO:90.
• the isolated nucleic acid molecule consists of SEQ ID NO:89 or its complement.
• the isolated nucleic acid molecule consists of SEQ ID NO:49 or its complement, SEQ ID NO:90.
• the present invention provides an isolated nucleic acid molecule that has a sequence identity of at least about 70% over the 548 bp region of SEQ ID NO:49. In preferred embodiments, the sequence identity is at least about 80%, preferably at least about 90%, more preferably at least about 95%. [14] In some embodiments, the present invention provides a probe comprising an isolated nucleic acid molecule of the present invention and a label.
• the present invention provides a probe comprising an isolated nucleic acid molecule of the present invention, a reporter molecule, and a quencher molecule.
• the reporter molecule produces a signal upon the separation of the reporter molecule and the quencher molecule.
• the quencher molecule is capable of quenching the signal of the reporter molecule.
• the reporter molecule is a fluorophore such as FAM, ROX, Texas Red, TET, TAMRA, JOE, HEX, CAL Red, and VIC, preferably the fluorophore is FAM, ROX, or Texas Red.
• the probe is capable of being cleaved by a protein thereby separating the reporter molecule from the quencher molecule.
• the protein is Taq polymerase.
• the present invention provides an assay which comprises using a probe of the present invention.
• the assay is a nucleic acid hybridization assay such as a TaqMan® based assay.
• the assay further comprises conducting PCR amplification.
• the assay may further comprise detecting the presence or measuring the amount of the probe and detecting the presence or measuring the amount of a target nucleic acid molecule.
• the absence of the target nucleic acid molecule and the absence of the probe indicate a false negative result for the target nucleic acid molecule and the absence of the target nucleic acid molecule and the presence of the probe indicate a true negative result for the target nucleic acid molecule.
• the present invention provides a kit for a probe-based nucleic acid assay comprising an isolated nucleic acid molecule of the present invention packaged with instructions for use.
• the isolated nucleic acid molecule contains a label such as a reporter molecule and a quencher molecule
• the probe-based nucleic acid assay is for the detection of an organism such as one belonging to Bacillus, Mycobacterium, Francisella, Brucella, Clostridium, Yersinia, Variola, Orthopox, or Burkholderia.
• the kit of the present invention may further include reagents or components for detecting the presence of a nucleic acid molecule belonging to the organism.
• the present invention also provides a method of making an internal positive control nucleic acid molecule for a probe-based nucleic acid molecule assay which comprises creating a first DNA fragment and a second DNA fragment from a template DNA and first set of primers and a second set of primers; creating a third DNA fragment and a fourth DNA fragment from the first DNA fragment and the second DNA fragment with a third set of primers and a second set of primers; hybridizing the third DNA fragment and the fourth DNA fragment to obtain a first hybridized DNA; using a fifth primer set to create a fifth DNA fragment from the first hybridized DNA; using a sixth primer set and a seventh primer set to create a sixth DNA fragment and a seventh DNA fragment from the fifth DNA fragment; creating a eighth DNA fragment and a ninth DNA fragment from the sixth DNA fragment and the seventh DNA fragment using a eighth primer set and a ninth primer set; hybridizing the eighth DNA fragment and the ninth DNA fragment to obtain a second hybridized DNA; creating a tenth DNA fragment and an eleventh DNA fragment
• the method of making an internal positive control nucleic acid molecule for a probe-based nucleic acid molecule assay wherein the internal positive control nucleic acid molecule contains a sequence that has a sequence identity of at least about 70% over the 548 bp region of SEQ ID NO:49.
• the sequence identity is at least about 80%, preferably at least about 90%, more preferably at least about 95%.
• Figure 1 is a schematic showing an example of a TaqMan® based assay.
• Figure 2 schematically shows the site-directed mutagenesis process used to generate the IPC nucleic acid molecule of the present invention.
• Figure 3 shows the gel of Example 4B.
• Figure 4 shows an agarose gel electrophoresis of denatured in vitro transcribed
• FIG. 5 shows DMSO is completely inhibitory at 25% and was partially inhibitory at 6.25%.
• Figure 6 shows guanidine hydrochloride was completely inhibitory at 200 mM and 100 mM.
• Figure 7 shows guanidine thiocyanate was completely inhibitory at 100 mM and
• FIG. 9 shows SDS was completely inhibitory at 0.02% and 0.01%.
• Figure 10 shows glycerol was completely inhibitory at 25%, 12.5%, and 6.25%.
• Figure 11 shows formamide was completely inhibitory at 25%, 12.5%, 6.25%, and 3.125%.
• Figure 12 shows EDTA was completely inhibitory at 5 mM and 2.5 mM.
• the present invention provides an internal positive control (IPC) for use in nucleic acid hybridization assays, preferably probe-based nucleic acid assays such as TaqMan® based assays.
• IPC internal positive control
• An example of a TaqMan® based assay is schematically shown in Figure 1.
• the present invention provides an oligonucleotide (IPC oligonucleotide) having a reporter molecule and a quencher molecule.
• the IPC oligonucleotide specifically anneals between the forward and reverse primers of a target sequence.
• the IPC oligonucleotide is cleaved by the 5' nuclease activity of Taq polymerase during PCR amplification and the reporter molecule is then separated from the quencher molecule to generate a sequence specific signal. With each amplification cycle, additional reporter molecules are separated from the quencher molecules.
• the intensity of a signal such as fluorescence, may be monitored before, during, or after PCR amplification or a combination thereof.
• the IPC nucleic acid molecule of the present invention may be used to distinguish a true negative result from a false negative result.
• a "true negative” result correctly indicates that a sample lacks a target nucleic acid sequence.
• a "false negative” result incorrectly indicates the absence of a target nucleic acid sequence which may result from PCR inhibitors present in the sample or technical error.
• the IPC nucleic acid molecule of the present invention may be used as a universal internal control as it comprises unique primer and probe sites and does not exhibit homology with any known nucleic acid sequences that may interfere with this assay, i.e. does not anneal with known nucleic acid sequences during conventional PCR techniques.
• the IPC nucleic acid molecule of the present invention provides greater flexibility over commercially available IPCs as a variety of reporter molecule and quencher molecule pairs may be used and since the primers, probes, and IPC nucleic acid molecule sequences are independent, various concentrations of each may be used.
• PA Protective Antigen
• AAAAGCGAAG TACAAGTGCT GGACCTACGG TTCCAGACCG TGACAATGAT GGA 3 ' (SEQ ID NO : 1) .
• the probe site was mutated first, followed by the upper primer site and then finally the lower primer site.
• the mutations were conducted with PCR-based site directed mutagenesis methods known in the art. See Courtney, B.C., et al. (1999) Analytical Biochemistry 270:249-256. The methods were the same for all three sites, the only differences were the mutagenic oligonucleotide sequences.
• mutations of each site were performed in three stages comprising five steps.
• genomic Bacillus anthracis DNA was used, and for the subsequent primer mutations, the plasmid DNA from the clone of the previous mutation was used.
• Mutagenic oligonucleotides were used to introduce the desired mutations.
• these oligos contained 1 A the sequence of B. anthracis genomic DNA and 1 A the sequence of the desired mutation.
• These mutagenic oligos were paired up with an oligo outside of the final 153 bp PA product. When amplified with PCR, the result was two products each containing half of the final desired mutation sequence.
• these mutagenic oligos consisted of 1 A the new sequence that was introduced in round 1 and 1 A the sequence of the rest of the desired mutation.
• the two products from round 1 were used as templates.
• these mutagenic oligos were paired up with an oligo outside of the final 153 bp PA product.
• the result was two products each containing all of the final desired mutation sequence.
• the two products from round 2 were used as primers on each other and ligated together, in addition the two oligos outside of the 153 bp product were used to further amplify it and increase the copy number of the final product.
• This final product was ligated into the pCR2.1 vector (Invitrogen Corporation, Carlsbad, CA) and transformed into competent INVaF' E. coli (Invitrogen Corporation, Carlsbad, CA).
• the template DNA used was 1 ng of Ames genomic DNA (USAMRIID, Ft. Detrick, MD), and the primers were: BANPAISl
• the product, Fragment 1 was a 252 bp product as follows: 5' GTAACAATGTGGGTAGATGACCAAGAAGTGATTAATAAAGCTTCTAATTCTAACAAAATCA GATTAGAAAAAGGAAGATTATATCAAATAAAAATTCAATATCAACGAGA ⁇ A ⁇ TCCTACTGAAA AAGGATTGGATTTCAAGTTGTACTGGACCGATTCTCAAAATAAAAAAGAAGTGATTTCTAGTG ATAACTTACAATTGCCAGAATTAAAACAAAAATCTTCGAACTCAAGAAAAACGGATGGACTGA GA 3' (SEQ ID NO: 10) .
• thetemplateDNAused was 1 ng of Ames genomic DNA (USAMRIID, Ft. Derrick, MD), andthe primerswere: BANPAIAl
• Theproduct, Fragment2 was a225 bpproduct as follows:
• the template DNA used was Fragment 1 .
• thetemplateDNAused was Fragment2
• the 225 bp productfrom BANPAIA1/PA35PCIU, andtheprimersused were: BANPAIAl
• Fragment 3 (265 bp product from BANPAISl /P A35PC2L) was hybridized with Fragment 4 (240 bp product from BANPAIAl /P A35PC2U).
• An additional l ⁇ M each of BANPAISl and BANPAIAl primers were added to create more product.
• a new primer set that was inside BANPAISl /BANPAIAl, but still outside BAPA3U/BAPA5L was developed.
• the primers set is as follows: BANPABISl
• Fragment 5 was 348 bp product and is as follows:
• Fragment 5 was gel purified with the QIAquick Gel Extraction Kit and cloned using the Original TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA). All of the clones were sequenced with both forward and reverse primers in duplicate. Clone 11 was chosen because it had the exact mutated sequence that we were trying to achieve and the rest of the sequence remained unaltered.
• the Qiagen Plasmid Mini Purification Kit (Qiagen, Carlsbad, CA) was used to purify the plasmid DNA for further mutations.
• the template DNA used was 1 ng of purified plasma DNA from Clone 11 having the following sequence: 5' GAAACAGCTATGACCATGATTACGCCAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGG CCGCCAGTGTGCTGGAATTCGGCTTCAACGAAAATCCTACTGAAAAAGGATTGGATTTCAA GTTGTACTGGACCGATTCTCAAAATAAAAAAGAAGTGATTTCTAGTGATAACTTACAATTGCC AGAATTAAAACAAAAATCTTCGAACTCAAGAAAA ⁇ CGGATGGACTGAGAGCGATCAACTACGT TCCAGACCGTGACAATGATGGAATCCCTGATTCATTAGAGGTAGAAGGATATACGGTTGATGT CAAAAATAAAAGAACTTTTCTTTCACCATGGATTTCTAATATTCATGAAAAGAAAGGATTAAC CAAATATAAATCATCTCCCGA ⁇ AAATGGAGCACGGCTTCTGATCCGTACAGTGATTTCAAGCC GAATTCT
• the template DNA used was 1 ng of purified plasma DNA from Clone 11 and the primers used were as follows: MOD31U
• Fragment 6 and Fragment 7 were purified with the QIAquick PCR Purification
• the product, Fragment 8 is a 147 bp product as follows:
• Fragment 8 and Fragment 9 were purified with the QIAquick Gel Extraction Kit
• the template DNA used was 1 ng of the purified plasmid DNA from clone 7.
• the primers used were as follows:
• thetemplateused was 1 ng ofthe purified plasmid DNA from clone 7, and the primers used were as follows:
• Kit (Qiagen, Valencia, CA).
• the template used was Fragment 10, the 262 bp product from M0D51U/PCR II FOR.
• the primers used were as follows: MOD52U
• TTCCGCATACCAGTTGTTGTCG 3' (SEQ ID NO: 45) .
• Kit (Qiagen, Valencia, CA).
• Fragment 12 was hybridized with
• SEQ ID NO:49 The underlined sequence in SEQ ID NO:49 indicates the 153 base amplicon sequence which is herein designated SEQ ID NO:89.
• the product was gel purified with the QIAquick Gel Extraction Kit and cloned using the Original TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA). All of the clones were sequenced with both forward and reverse primers in duplicate. Clone 1 was selected because it had the mutated sequence of the 548 bp product (SEQ ID NO:49) and the rest of the sequence remained unaltered.
• the Qiagen Plasmid Mini Purification Kit (Qiagen, Carlsbad, CA) was used to purify the plasmid DNA, the final clone.
• the mutated sequences were identical to the wild type sequences in length, base composition and location. Mutated sequences were chosen that had optimal primer characteristics without matching any known naturally occurring DNA sequences. The mutated sequences were checked with a nucleotide BLAST search to confirm the uniqueness of the sequences. See http://www.ncbi.nlm.nih.gov/BLAST/. The final product is the IPC nucleic acid molecule of the present invention, a cloned fragment of DNA having a unique sequence that can be used in any probe-based PCR assay with specific primers and probes.
• the IPC nucleic acid molecule of the present invention may be used with fluorescence resonance energy transfer (FRET), Scorpions, and Molecular Beacons assays. See Szollosi, etal. (1998) Cytometry 34(4): 159-179; Schweitzer and Kingsmore (2001) Curr. Opin. Biotechnol. 12(l):21-27; and Antony and Subramaniam (2001) J. Biomol. Struct. Dyn. 19(3):497-504, which are herein incorporated by reference.
• FRET fluorescence resonance energy transfer
• the IPC nucleic acid molecule of the present invention may be used to develop an internal positive control ribonucleic acid molecule for reverse-transcriptase PCR reactions.
• the IPC nucleic acid molecules for use in reverse transcriptase PCR applications are herein referred to as "RT-IPC nucleic acid molecules".
• the RT-IPC nucleic acid molecule was sensitive to a variety of known reverse transcriptase inhibitors, as well as, several PCR inhibitors, including heparin, EDTA, glycerol, guanidine thiocyanate, DMSO, SDS, guanidine hydrochloride, and formamide.
• the RT-IPC nucleic acid molecule of the present invention is may be used to monitor inhibitors of RT-PCR applications and build confidence in negative results obtained with agent specific assays.
• Example 4 As provided in Example 4, purified IPC plasmid DNA was linearized with the restriction enzyme Spel. The T7 promoter on the pCR 2.1 vector (which contains the IPC DNA insert) was then used to in vitro transcribe the IPC RNA, using methods known in the art. The resultant IPC RNA, RT-IPC nucleic acid molecule, can be used in reverse transcription PCR (RT-PCR) assays to monitor inhibition in the same capacity that the IPC nucleic acid molecule of the present invention is used for PCR assays.
• RT-PCR reverse transcription PCR
• RT-PCR of RNA templates uses two enzymes, reverse transcriptase and Taq
• RT-PCR thus has two weak points at which inhibition can occur, making it even more susceptible to inhibition than PCR.
• the RT-IPC nucleic acid molecule may be used to detect inhibitors of Taq polymerase as well as inhibitors of reverse transcriptase.
• the RT-IPC nucleic acid molecule of the present invention includes RNase- resistant RT-IPC nucleic acid molecules as known in the art.
• RNase-resistant nucleic acid molecules include complexes of MS2 bacteriophage coat proteins and RNA molecules produced in Escherichia coli by the induction of an expression plasmid that encodes the coat protein and the RNA sequence.
• the RNA sequences are protected from RNase digestion within the bacteriophage-like complexes (Ambion, Inc., Austin, TX).
• RNase-resistant RT-IPC nucleic acid molecules according to the present invention allow use of the RT-IPC nucleic acid molecules throughout an entire assay procedure, i.e. from extraction of the RNA to amplification.
• RNase-resistant RT-IPC nucleic acid molecules according to the present invention have greater stability. See Eisler, D. L. et al. (2004) J. Clin. Microbiol. 42(2):841-844; Bressler, A.M. and Nolte, F.S. (2004) J. Clin Microbiol. 42(3):987-491; BeId, M. et al. (2004) J. Clin. Microbiol. 42(7):3059- 3064, which are herein incorporated by reference.
• the IPC and RT-IPC nucleic acid molecules of the present invention may be used in a probe-based nucleic acid diagnostic assay to determine the presence or absence of PCR inhibitors.
• An assay utilizing the IPC and RT-IPC nucleic acid molecules of the present invention can either be run by itself or multiplexed with any other diagnostic assay.
• the IPC and RT-IPC nucleic acid molecules of the present invention may be used as a control assay to trouble-shoot probe-based nucleic acid assay problems such as PCR assays.
• the IPC and RT-IPC nucleic acid molecules of the present invention may be used to determine if a problem relates to the reagents, operator technique, or instrumentation.
• the IPC nucleic acid molecule, RT-IPC nucleic acid molecule, or both may be multiplexed or used in conjunction with other assays for the detection of an organism based on the presence of a target nucleic acid molecule that is unique to the organism.
• the IPC and RT-IPC nucleic acid molecules of the present invention may be used in conjunction with assays, known in the art, for organisms belonging to Bacillus, Mycobacterium, Francisella, Brucella, Clostridium, Yersinia, Variola, Orthopox, and Burkholderia. See e.g. Fasanella, A. et al. (2003) J. Clin. Microbiol.
• nucleic acid molecule As used herein, “nucleic acid molecule”, “polynucleotide”, and “oligonucleotide” are used interchangeably to refer DNA and RNA molecules of natural or synthetic origin which may be single-stranded or double-stranded, and represent the sense or antisense strand.
• the nucleic acid molecules of the present invention may contain known nucleotide analogs or modified backbone residues or linkages, and any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
• the IPC and RT-IPC nucleic acid molecules of the present invention are isolated.
• isolated refers to a nucleic acid molecule that is isolated from its native environment.
• An “isolated” nucleic acid molecule may be substantially isolated or purified from the genomic DNA of the species from which the nucleic acid molecule was obtained.
• An “isolated” polynucleotide may include a nucleic acid molecule that is separated from other DNA segments with which the nucleic acid molecule is normally or natively associated with at either the 5' end, 3' end, or both.
• the IPC and RT-IPC nucleic acid molecules of the present invention may be in their native form or synthetically modified.
• the IPC and RT-IPC nucleic acid molecules of the present invention may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
• RNA molecules include mRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns.
• the IPC and RT-IPC nucleic acid molecules of the present invention may be linked to other nucleic acid molecules, support materials, reporter molecules, quencher molecules, or a combination thereof.
• nucleic acid molecules include promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA or PCR protocol. In some embodiments of the present invention, nucleic acid sequences comprising the IPC oligonucleotide described herein are contemplated.
• the IPC and RT-IPC nucleic acid molecules of the present invention may be readily prepared by conventional methods known in the art, for example, directly synthesizing the nucleic acid sequence using methods and equipment known in the art such as automated oligonucleotide synthesizers, PCR technology, recombinant DNA techniques, and the like.
• the IPC and RT-IPC nucleic acid molecules of the present invention may contain a label such as quencher molecule and a reporter molecule.
• a label such as quencher molecule and a reporter molecule.
• a wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays employing the IPC and RT-IPC nucleic acid molecules of the present invention.
• a "label” or a "detectable moiety” is a composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
• a "labeled" nucleic acid molecule comprises a bound label such that the presence of the nucleic acid molecule may be detected by detecting the presence of the label bound to thereto.
• the label may be bound to the nucleic acid molecule via a covalent bond, such as a chemical bond, or a noncovalent bond, such as ionic, van der Waals, electrostatic, or hydrogen bonds.
• a covalent bond such as a chemical bond
• a noncovalent bond such as ionic, van der Waals, electrostatic, or hydrogen bonds.
• Suitable reporter molecules and quencher molecules include radionucleotides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
• a fluorescent reporter molecule and quencher molecule are used.
• nucleic acid probe and “probe” refers to a nucleic acid molecule that is capable of binding to a target nucleic acid molecule having a sequence that is complementary to the sequence of the nucleic acid probe.
• a probe may include natural or modified bases. See e.g. MPEP 2422, 8 th ed., which is herein incorporated by reference.
• the nucleotide bases of the probe may be joined by a linkage other than a phosphodiester bond, so long as the linkage does not interfere with the ability of the nucleic acid molecule to bind a complementary nucleic acid molecule.
• the probe may bind a target sequence that is less than 100% complementary to the probe sequence and such binding depends upon the stringency of the hybridization conditions.
• the presence or absence of the probe may be detected to determine the presence or absence of a target sequence or subsequence in a sample.
• the probe may contain a label whose signal is detectable by methods known in the art.
• a "signal" is a measurable characteristic.
• the label is a reporter molecule and a quencher molecule
• the signal may increase or decrease upon dissociation of reporter molecule and the quencher molecule. For example, if the reporter molecule is a fluorophore, separation of the quencher from the fluorophore will generate a detectable signal due to an increase in light energy emitted by the fluorophore in response to illumination.
• a "target" nucleic acid molecule may be any nucleic acid molecule, the presence and/or amount of which is desired to be known.
• the sequence of the target nucleic acid molecule is known.
• the sequence of the target nucleic acid molecule may be a sequence that is suspected of having alterations, i.e. differences, from a reference nucleic acid sequence.
• the sequence of the target nucleic acid molecule may or may not be known, and the "reference nucleic acid sequence" is a known nucleic acid sequence to which the sequence of the target nucleic acid molecule may be compared.
• the alteration in the target nucleic acid molecule may be in a single nucleotide base or more than a single nucleotide base.
• Such an alteration may be a known polymorphic alteration, such as a single nucleotide polymorphism.
• kits for use with nucleic acid hybridization assays such as PCR amplification and PCR assays, including TaqMan® based assays, fluorescence resonance energy transfer (FRET), Scorpions, and Molecular Beacons assays. See Szollosi, et al. (1998) Cytometry 34(4):159-179; Schweitzer and Kingsmore (2001) Curr. Opin. Biotechnol. 12(l):21-27; and Antony and Subramaniam (2001) J. Biomol. Struct. Dyn. 19(3):497-504, which are herein incorporated by reference.
• kits comprise the IPC nucleic acid molecule, RT-IPC nucleic acid molecule, or both, and one or more components necessary for performing the assay. Components may be compounds, reagents, containers, instructions and/or equipment.
• kits may be used for any one or more of the uses described herein, and, accordingly, may contain instructions for any one or more of the following uses: determining whether a target nucleic acid sequence is present in a sample, detecting a target nucleic acid sequence, quantifying a target nucleic acid sequence, comparing target nucleic acid sequence to a reference sequence, determining genotype, determining allele composition of a target nucleic acid, detecting and/or quantifying multiple nucleic acid sequences, and use of the methods in conjunction with nucleic acid amplification techniques.
• kits of the invention comprise one or more containers comprising any combination of the components or reagents described herein.
• the kit comprises the IPC nucleic acid molecule, RT-IPC nucleic acid molecule, or both and a set of primers and probes for conducting an assay for a target nucleic acid molecule.
• the kit may further include at least one label and at least one substrate or for producing a signal.
• the kit may further include deoxynucleoside triphosphates and/or ribonucleoside triphosphates.
• the kit may further include one or more suitable buffers for conducting the given assay.
• Each component of the kit can be packaged in separate containers or some components can be combined in one container where cross-reactivity and shelf life permit.
• kits of the invention may optionally include a set of instructions, generally written instructions, although electronic storage media (e.g., magnetic diskette or optical disk) containing instructions are also acceptable, relating to the use of components of the methods of the present invention for the intended nucleic acid detection and/or quantification, and/or, as appropriate, for using the detection and quantification methods in conjunction with amplification techniques.
• the instructions included with the kit generally include information as to reagents (whether included or not in the kit) necessary for practicing the methods of the presentation invention, instructions on how to use the kit, and/or appropriate reaction conditions.
• sequence identity in the context of two or more nucleic acid molecules, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotide bases that are the same (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
• the percentage of sequence identity may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• BLAST and BLAST 2.0 algorithms may be used to determine the sequence identity of two or more sequences. See Altschul et a (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. MoL Biol. 215:403-410, which are herein incorporated by reference. BLAST analyses are publicly available through the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.
• the IPC nucleic acid molecules of the present invention include nucleic acid molecules that have at least about 70% identity, preferably about 80% identity or more, more preferably about 90% identity or more, more preferably about 95% identity or more, over the 548 bp region set forth in SEQ ID NO:49.
• Nucleic acid molecules that have sequences that have at least about 70% identity to SEQ ID NO:49 are "substantially identical" to SEQ ID NO:49.
• a plasmid containing the IPC nucleic acid molecule of the present invention was used to obtain an IPC RNA having SEQ ID NO:90, which is the complementary sequence from nucleotide position 55 to 508 of SEQ ID NO:49.
• SEQ ID NO: 90 contains the RT-IPC amplicon which is underlined and is herein designated as SEQ ID NO:91, which is the complementary sequence from nucleotide position 105 to 257 of SEQ ID NO:89.
• the reverse transcription-PCR target within SEQ ID NO: 90 begins at nucleotide position 105 and ends at nucleotide 257.
• the RT-IPC nucleic acid molecules of the present invention include nucleic acid molecules that have at least about 70% identity, preferably about 80% identity or more, more preferably about 90% identity or more, more preferably about 95% identity or more, over the 153 base region of SEQ ID NO:90.
• the RT-IPC nucleic acid molecules of the present invention include nucleic acid molecules that have at least about 70% identity, preferably about 80% identity or more, more preferably about 90% identity or more, more preferably about 95% identity or more, over the 453 base region of SEQ ID NO:90.
• the IPC and RT-IPC nucleic acid molecules of the present invention contains at least 80 consecutive bases of SEQ ID NO: 89 or its complement. In some embodiments, the IPC or RT-IPC nucleic acid molecules comprise SEQ ID NO:89 or its complement.
• Nucleic acid molecules that have sequences that have at least about 70% identity to a given sequence are “substantially identical" to the given sequence.
• the phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a nucleic acid molecule to a particular nucleotide sequence only in a sample comprising other nucleic acid molecules under stringent hybridization to moderate hybridization conditions.
• a positive signal is at least about 2 times, preferably about 5 times, more preferably about 10 times the background hybridization.
• Stringent hybridization conditions are about 5 0 C below the thermal melting temperature (Tm) of the probe to about 10 °C below Tm.
• Moderate hybridization conditions are about 10 0 C below the thermal melting temperature (Tm) of the probe to about 20 °C to about 25 0 C below Tm.
• Figure 2 schematically shows the site-directed mutagenesis process used to generate the IPC nucleic acid molecule of the present invention.
• the first two sets of PCR reactions allowed 1 A of the mutated sequence to be incorporated into the generated PCR fragments. Template DNA was placed in one reaction with primers 1 and 2, and another reaction with primers 3 and 4.
• the next two sets of PCR reactions allowed for full incorporation of the mutated sequence into the PCR products, hi one reaction, the primer set 1 and 2 generated DNA fragment was used as a template for amplification with primers 1 and 5.
• the other reaction used the primer set 3 and 4 generated DNA fragment as a template for amplification with primers 4 and 6.
• both fragments from primers 1 and 5 and from primers 4 and 6 were used as primers for each other in an overlap extension reaction. Partway through the PCR, primers 1 and 4 were added to the reaction. The final product was one DNA fragment that fully incorporated the mutated sequence.
• IPC nucleic acid molecule of the present invention The effect of three inhibitors, hemoglobin, heparin, and EDTA, on the IPC nucleic acid molecule of the present invention was tested.
• the IPC DNA was titrated to use the smallest amount possible to still obtain consistent results, yet make the assay very sensitive to inhibition.
• Smart Cycler® (Cepheid, Sunnyvale, CA) 1 fg of IPC DNA was found to be optimal for the methods herein.
• the reagents used were Idaho Technology PCR Reagents (Idaho Technology, Idaho Falls, Idaho), which include the 1OX buffer with 30 mM MgCl 2 and 1OX dNTP.
• 5X SC buffer an additive recommended by Cepheid (Cepheid, Sunnyvale, CA), the manufacturers of the Smart Cycler®, and Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA), was also used.
• the PCR was started with 2-minute activation at 95 0 C and then 45 cycles of 95 0 C for 1 second and 65 0 C for 20 seconds.
• the assay was tested against these three inhibitors using probes labeled with two different reporter dyes, FAM and ROX. The quencher on both of these probes was TAMRA. Biosearch Technologies, Novato, CA, manufactured the probes.
• EDTA was completely inhibitory at 5 mM and 2.5 mM. With the FAM probe, inhibition was completely relieved at 1.25 mM. With the ROX probe, 1.25 mM was partially inhibitory and inhibition was not completely relieved until 0.625 mM.
• the IPC nucleic acid molecule of the present invention affects the limit of detection of a given assay, the following was conducted. [119] The IPC nucleic acid molecule was duplexed with all of the assays that we currently use on the Smart Cycler®. The IPC nucleic acid molecule was labeled with ROX and the primary assay probes were labeled with FAM. Ten-fold serial dilutions were performed on all genomic DNA samples for the primary assays. The limit of detection was tested in triplicate for each assay.
• the primer set used was BAPA3U/5L and the probe was BAPA3P2A.
• BAP A5L b' TCCATCATTGTCACGGTCTGG 3' (SEQ ID NO: 51)
• BAPA3P2A
• the reaction mix included 1 OX PCR buffer with 5 mM MgCl 2 (Idaho
• the primer set used was BACAPBU2/L2 and the probe was BACAPBP2.
• the reaction mix included 1 OX PCR buffer with 5 mM MgCl 2 (Idaho
• the primer set used was BROMPF394/R474 and the probe was BROMP25-
• the reaction mix included 1 OX PCR buffer with 5 mM MgCl 2 (Idaho
• the primer set used was CBOTA4U/4L and the probe was CBOTA4P2A.
• the reaction mix included 1 OX PCR buffer with 4 mM MgCl 2 (Idaho).
• the primer set used was YPPLA3U/3L and the probe was YPPLAP3F.
• the reaction mix included 1 OX PCR buffer with 5 mM MgCl 2 (Idaho
• the primer set used was BACAPB4U/4L and the probe was BACAPBPlS.
• the reaction mix included 1 OX PCR buffer with 5 mM MgCl 2 (Idaho
• the primer set used was BAVRRA3U/3L and the probe was BAVRRA3P 1 S .
• the reaction mix included 1 OX PCR buffer with 3 mM MgCl 2 (Idaho
• the primer set used was FTTULU1/L1 and the probe was FTTULPlF.
• the reaction mix included 1 OX PCR buffer with 5 mM MgCl 2 (Idaho
• the primer set used was YPPIMU1/L 1 and the probe was YPPIMP 1 R.
• the reaction mix included 1 OX PCR buffer with 5 niM MgCl 2 (Idaho
• the primer set used was OPSPF89/R219 and the probe was Op-pl43S.
• the reaction mix included 1 OX PCR buffer with 5 mM MgCl 2 (Idaho
• the primer set used was FOPAF708/R846 and the probe was FtFOP A765S.
• the reaction mix included 1 OX PCR buffer with 5 niM MgCl 2 (Idaho
• the primer set used was J7R3U/3L and the probe was VARJ7R3p.
• VARJ7R3p 5 ' TCATCTGGAGAATCCACAACA 3 ' ( SEQ ID NO : 84 ) VARJ7R3p:
• the reaction mix included 1 OX PCR buffer with 5 mM MgCl 2 (Idaho
• the primer set used was BPISO2F1/R1 and the probe was BMISO2PF3.
• the reaction mix included 1 OX PCR buffer with 5 mM MgCl 2 (Idaho
• the plasmid containing the IPC nucleic acid molecule was linearized by restriction digest in order to provide a linear template for in vitro transcription of RNA. Briefly, 10 ⁇ g of IPC plasmid was cut in a restriction digest with 10 U of Spel restriction endonuclease (Invitrogen, Carlsbad, CA) in a restriction digest reaction in which the following reagents were added to a 1.7 ml microcentrifuge tube to a total volume of 40 ⁇ l:
• MMG molecular biology grade
• IPC plasmid DNA (Sephadex® G50 purified, amount of DNA about 10 ⁇ g)
• a 1% agrose gel was prepared by adding 0.5 g of molecular biology grade agarose (Invitrogen, Carlsbad, CA) to 50 ml of IX TAE buffer (Invitrogen, Carlsbad, CA) and microwaving for 2 minutes to dissolve the agarose.
• the agarose solution was poured into a gel casting template and a comb was inserted to form wells.
• the agarose solution was allowed to cool until a hardened gel formed.
• the gel was transferred to an electrophoresis box, and a solution of IX TAE (Invitrogen, Carlsbad, CA) containing 0.5 ⁇ g/ml of ethidium bromide was poured into the gel box until it covered the gel.
• G50 eluate of purified DNA prepared from an incomplete digestion), and plasmid linearized as described above were added to separate microcentifuge tubes.
• Various volumes of MBG water were added to each tube in order to bring the volume up in each tube to 9 ⁇ l.
• 1OX agarose gel loading buffer (Invitrogen, Carlsbad, CA) was added to each tube, and the tubes were mixed. The contents of each tube were added to separate wells of the 1% agarose gel.
• the gel electrophoresis box was connected to a power supply and the gel was electrophoresed at 125 volts until the dye present in the loading buffer had run three fourths the length of the gel.
• FIG. 1 shows the gel of Example 4B.
• Lane 1 is 3 ⁇ l (0.3 ⁇ g ) a 1 kb DNA ladder
• Lane 2 is 2 ⁇ l (0.2 ⁇ g) of a 1 :10 dilution of uncut plasmid DNA
• lane 3 is 0.5 ⁇ l (0.2 ⁇ g) of partially cut plasmid DNA that was purified by Sephadex® G50 purification
• lane 4 is 1 ⁇ l (0.25 ⁇ g) of plasmid DNA linearized according to Example 4A.
• the IPC RNA was transcribed from 1 ⁇ g of linear IPC by in vitro transcription using mMessage MachineTM with SuperRNAsinTM (Ambion, Austin, TX) at 37 0 C following all the manufacturers instructions except that all components of the reaction were doubled resulting in a final volume of 40 ⁇ l. Briefly, the following Ambion reaction components were assembled in a 1.7 ml RNase free microcentrifuge tube (Ambion, Austin, TX) as shown below to a final volume of 40 ⁇ l:
• the assembled reaction was mixed by gently flicking the bottom of the 1.7 ml microcentrifuge tube, and then placed in a 37 0 C incubator. After a 1 hour incubation, 1 ⁇ l of 2 U/ ⁇ l RNase-free DNase I (Ambion, Austin, TX) was added to the reaction. The microcentrifuge tube was mixed by gently flicking the bottom of the tube. The tube was centrifuged briefly then incubated at 37 0 C for 15 minutes.
• Qiagen RNeasy Mini Kit Qiagen, Valencia, CA
• the cap on the column was closed, and the column with collection tube were centrifuged for 15 seconds at 8,000 X g. The flow-through and collection tube were both discarded.
• the RNeasy column was placed into a new 2 ml collection tube, and 500 ⁇ l of Buffer RPE (Qiagen, Valencia, CA) was pipetted onto the RNeasy column. The tube was closed and centrifuged again as in the previous step. Again, the collection tube and flow- though were discarded. The Buffer RPE wash step was repeated again, and the column was inserted into a new collection tube. Next, 30 ⁇ l of RNase-free water was pippeted directly onto the silica-gel membrane in the RNeasy column.
• Buffer RPE Qiagen, Valencia, CA
• the cap was closed, and the column was centrifuged for 1 minute at 8,000 X g in order to elute the RNA.
• the elution step was repeated with a new collection tube and another 30 ⁇ l of RNase-free water.
• the eluates collected after elution 1 and elution 2 were designated eluate 1 and eluate 2, respectively.
• a 50 ml 1% agarose gel was prepared by adding 0.5 g of molecular biology grade agarose to 50 ml of 1 X TAE buffer and microwaving for 2 minutes to dissolve the agarose.
• the agarose solution was poured into a gel casting template and a comb was inserted to form wells.
• the agarose solution was allowed to cool until a hardened gel formed.
• the gel was transferred to an electrophoresis box, and a solution of IX TAE containing 0.5 ⁇ g/ml ethidium bromide was poured into the gel box until it covered the gel.
• RNA samples were mixed with 2X Gel Loading Buffer II (Ambion, Austin, TX) and heated at 65 0 C for 2 minutes to denature the RNA.
• Figure 4 shows an agarose gel electrophoresis of denatured in vitro transcribed IPC RNA.
• Lane 1 is an RNA ladder
• lane 2 is DNase treated transcription reaction
• lane 3 is RNeasy eluate 1
• lane 4 is RNeasy eluate 2.
• the agarose gel electrophoresis gel of Figure 4 shows a single band of RNA at the expected size for the DNase treated transcription reaction (lane 2), RNeasy eluate 1 (lane 3), and RNeasy eluate 2 (lane 4), respectively.
• the gel also shows that the concentration of RNA in the original sample (prior to Qiagen purification) was slightly higher than the first Qiagen eluate. The concentration of RNA in the second Qiagen eluate was much less than the first. In addition, the gel shows that there was no degraded RNA.
• RNA was quantified by measuring the OD 260 for duplicate samples comprising 2 ⁇ l of IPC RNA (RNeasy eluate) in total volume of 100 ⁇ l of RNase-free TE pH 8.0 (Quality Biological, Gaithersburg, MD). The absorbance values were averaged and used to calculate the concentration of RNA using the formula: average absorbance at 260 nm x dilution factor x extinction coefficient concentration of RNA; where the extinction coefficient for single stranded RNA was 40 ⁇ g RNA/ml. By this method the concentration of RNA was determined to be 0.169 ⁇ g/ ⁇ l.
• RNA transcribed was 5.1 ⁇ g (0.169 ⁇ g/ ⁇ l x 30 ⁇ l), which is sufficient for a large number of reverse transcription and PCR reactions.
• the RT-IPC nucleic acid molecule was amplified with IPC 5U and IPC 3L primers using methods known in the art.
• the RT-PCR kit used for all verification assays was Super-ScriptTM One-Step RT-PCR with Platinum® Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA). All verification experiments were performed on R.A.P.I.D. ® (Idaho Technology, Inc., Salt Lake City, UT).
• the RT-IPC nucleic acid molecule was titrated to use the smallest amount possible to still obtain consistent results, yet make the assay very sensitive to inhibition. [162] Using the R.A.P.I.D . ® (Idaho Technology, Inc., Salt Lake City, UT) 1 fg of IPC
• RNA was found to be optimal for the methods herein.
• the reagents used were from the Super- ScriptTM One-Step RT-PCR Kit with Platinum® Taq DNA Polymerase (Invitrogen Life Technologies, Carlsbad, CA), which include the 2X reaction mix and 50 mM MgSO 4 .
• the RT-PCR was started with a reverse transcriptase incubation at 50 °C for 15 minutes, followed by a 5 -minute reverse transcriptase deactivation/Taq polymerase activation at 95 °C and then 45 cycles of 95 0 C for 1 second and 65 °C for 20 seconds.
• the assay was tested against these eight inhibitors using probes labeled with a reporter dye, FAM.
• the quencher on the probe was TAMRA. Biosearch Technologies, Novato, CA, manufactured the probes.

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