Claims for Patent: 8,940,487

Summary for Patent: 8,940,487

Title:	Methods and compositions for universal detection of nucleic acids
Abstract:	Provided are methods and compositions for detecting the presence or amount of one or more target nucleic acids in a sample. Methods of the present invention include linking universal nucleic acid segments into a single molecule in a linking reaction dependent on a target nucleic acid of interest. A variety of universal segment linking strategies are provided, including preamplification by polymerase chain reaction, ligation-based strategies, reverse transcription and linear polymerase extension. Linking the universal segments into a single molecule generates a tagged target nucleic acid which is detected in a manner dependent on an intramolecular interaction between one universal segment and a second portion of the tagged target nucleic acid. In certain embodiments, the intramolecular interaction includes the formation of a hairpin having a stem between a universal segment at one end of the tagged target nucleic acid and a second universal segment at the opposite end of the tagged target nucleic acid. A variety of detection formats are provided, including solution-phase and surface-based formats. The methods and compositions are well-suited for highly multiplexed nucleic acid detection, and are applicable for the detection of any target nucleic acid of interest in both research and clinical settings.
Inventor(s):	Spier; Eugene (Los Altos, CA)
Assignee:	UmiTaq Bio (Los Altos, CA)
Application Number:	12/931,803
Patent Claims:	1. A method of detecting the presence or amount of a first target nucleic acid in a sample, the method comprising: (a) linking first and second universal DNA segments into a first molecule in a linking reaction dependent on the first target nucleic acid, thereby providing a first tagged target nucleic acid; and (b) PCR amplifying the first tagged target nucleic acid using first and second universal primers, each universal primer having a 3' portion that anneals to one of the two universal DNA segments; wherein: the 3' portion of the first universal primer anneals to the first universal segment; a 5' portion of the first universal primer comprises a nucleic acid sequence sufficiently similar to a portion of the first universal DNA segment, a portion of the second universal DNA segment, or a portion of the first target nucleic acid, to anneal to a complement of the portion of the first universal DNA segment, a complement of the portion of the second universal DNA segment, or a complement of the portion of the first target nucleic acid, respectively, under PCR reaction conditions; an amplicon generated upon extension of the first universal primer forms an intramolecular hairpin stem between the 5' portion of the first universal primer and a portion of the amplicon complementary to the first universal DNA segment, the second universal DNA segment, or the first target nucleic acid; or wherein the first universal primer forms a circular structure upon annealing to the first tagged nucleic acid that brings the 3' and 5'-ends of the first universal primer close to each other; and, formation of the hairpin stem or circular structure results or causes a change in a first detectable signal, the first detectable signal indicating the presence or quantity of the first target nucleic acid in the sample. 2. The method of claim 1, wherein the first target nucleic acid comprises a nucleic acid or nucleic acid feature selected from: a DNA, an RNA, a bisulphite treated DNA, a mammalian nucleic acid, a primate nucleic acid, a rodent nucleic acid, a viral nucleic acid, a bacterial nucleic acid, an archaea nucleic acid, a cDNA, a cDNA corresponding to a short RNA, a genetic variant, a mutation or insertion that confers drug resistance, a somatic mutation, a polymorphism, a single nucleotide polymorphism, a rare allele, a portion of the KRAS gene, a nucleic acid that exhibits a variation in copy number, an intron, an exon, an intron-exon boundary, a splice junction, one or more dinucleotides corresponding to one or more methylated or unmethylated CpG dinucleotides, a nucleic acid comprising one or more restriction enzyme recognition sequences, a portion of human chromosome 21, a portion of the human X chromosome, and a portion of the human Y chromosome. 3. The method of claim 1, wherein linking first and second universal DNA segments into a first molecule in a linking reaction dependent on the first target nucleic acid comprises performing a reaction selected from: (a) a PCR reaction, the reaction comprising: a first primer having a 3' portion that anneals to a first portion of the first target nucleic acid and a 5' portion comprising the first universal DNA segment; and a second primer having a 3' portion that anneals to a second portion of the first target nucleic acid and a 5' portion comprising the second universal DNA segment; wherein amplification of the first target nucleic acid using the first and second primers links the first and second universal DNA segments into a single molecule; (b) a ligation reaction, wherein oligonucleotides complementary to the first target nucleic acid comprise 5' and 3' ends extending beyond the first target nucleic acid, and wherein the oligonucleotides serve as a template to ligate the first and second universal DNA segment to the first target nucleic acid; (c) a ligation reaction, wherein the first universal DNA segment is ligated to the second universal DNA segment on the first target nucleic acid, thereby linking the first and second universal DNA segments together into a single molecule; (d) a reverse transcription reaction, the reaction comprising: generating a first cDNA strand by extending a first primer having a 3' portion that anneals to a first portion of the first target nucleic acid and a 5' portion comprising the first universal DNA segment, wherein the target nucleic acid is an RNA; and generating a second cDNA strand by extending a second primer having a 3' portion that anneals to a portion of the first cDNA and a 5' portion comprising the second universal DNA segment; wherein synthesis of the first and second cDNA strands links the first and second universal DNA segments into a single molecule; or (e) a linear polymerase extension reaction, wherein the first universal DNA segment is linked to the first target nucleic acid by a polymerase that extends on the template complementary to the first universal DNA segment, and wherein a primer with a 5' tail comprising the second universal DNA segment introduces the second universal DNA segment, thereby linking the first and second universal segments into a single molecule. 4. The method of claim 1, wherein step (a) is multiplexed such that multiple target nucleic acids of different nucleic acid sequences are tagged with the same or different pairs of universal DNA segments. 5. The method of claim 1, wherein step (a) further comprises linking one or more additional pairs of universal DNA segments into one or more additional molecules in a linking reaction specific to one or more additional target nucleic acids, thereby providing one or more additional tagged target nucleic acids in a single reaction volume. 6. The method of claim 1, wherein step (a) further comprises linking third and fourth universal DNA segments into a second molecule in a linking reaction specific to a second target nucleic acid, thereby providing a second tagged target nucleic acid. 7. The method of claim 6, wherein the second tagged target nucleic acid is PCR amplified using universal primers specific to the third and fourth universal DNA segments, and wherein amplicons generated from amplification of the second tagged target nucleic acid form hairpin stems or circles that result in a second detectable signal that is distinguishable from the first detectable signal. 8. The method of claim 1, wherein step (a) occurs in a first reaction location and step (b) occurs in one or more different reaction locations. 9. The method of claim 8, wherein the one or more reaction locations of step (b) comprise a well, a nano-well, a droplet, an array structure, a flat surface or a bead surface. 10. The method of claim 9, wherein the one or more reaction locations comprise an array structure or a bead surface, and wherein the first universal primer is attached to the array structure, the flat surface or the bead surface. 11. The method of claim 8, wherein between steps (a) and (b), a reaction mixture comprising the product of the linking reaction at the first location is transferred to the one or more different locations, wherein the reaction mixture is optionally diluted prior to, or during, transfer. 12. The method of claim 11, wherein the first and second universal primers are delivered to the one or more different reaction locations, and wherein the first and second universal primers are delivered prior to, during, or after the linking reaction mixture is transferred to the one or more different reaction locations. 13. The method of claim 1, wherein the first universal primer comprises a first label proximal to one end of the 5' portion of the first universal primer. 14. The method of claim 13, wherein the first label comprises a fluorescent dye. 15. The method of claim 13, wherein the first universal primer comprises a label quencher or FRET dye disposed proximal to the same end or proximal to an end opposite the 5' portion of the first universal primer as compared to the first label, and wherein the label quencher or FRET dye is disposed at an effective quenching or FRET distance from the first label. 16. The method of claim 15, wherein the first detectable signal resulting from hairpin stem or circle formation comprises a change of FRET caused by changing the distance between the first label and the label quencher or FRET dye. 17. The method of claim 16, wherein changing the distance between the first label and the label quencher or FRET dye is performed by a mechanism selected from: (a) removal of the label and/or label quencher or FRET dye from the amplicon or primer by a nuclease; (b) increasing the distance between the first label and the label quencher or FRET dye, wherein the label quencher or FRET dye is initially disposed at an effective quenching or FRET distance from the first label via a double-stranded hairpin stem or a random coil ssDNA structure in the first universal primer, and wherein the distance between the label quencher or FRET dye and the first label is increased by the intramolecular hairpin that is formed within the amplicon generated upon extension of the first universal primer; (c) melting of an oligonucleotide from the first universal primer, wherein the oligonucleotide comprises the label quencher or FRET dye, wherein the first universal primer comprises the first label, and wherein the hairpin stem or circle generated upon extension of the first universal primer melts the oligonucleotide from the first universal primer, thereby increasing the distance between the label quencher or FRET dye and the first label; (d) forming a hairpin stem or circle disposes the first label at an effective FRET distance from a quencher or FRET dye attached to an oligonucleotide that is complementary to the amplicon generated upon extension of the first universal primer. 18. The method of claim 17, wherein the first label is disposed between the 3' and 5' portions of the first universal primer, wherein the label quencher or FRET dye are disposed at the 5' portion of the first universal primer as compared to the first label, wherein the label quencher is removed from the amplicon with the nuclease, and wherein the labeled amplicon is detected by capillary electrophoresis or hybridization to a surface-bound oligonucleotide comprising a region complementary to the amplicon. 19. The method of claim 15, wherein the label quencher or FRET dye is disposed at a position on the first universal primer selected from: a position between the 5' and 3' portions of the first universal primer, and a position 5' of the junction between the 5' and 3' portions of the first universal primer. 20. The method of claim 1, wherein the first universal primer comprises a polymerase blocking unit disposed between the 5' and 3' portions of the first universal primer. 21. The method of claim 1, wherein the different pairs of universal primers are labeled with different dyes, the different dyes having different emission wavelengths, and wherein during step (b), the different dyes having different emission wavelengths are detected separately. 22. The method of claim 4, wherein the target nucleic acids of different nucleic acid sequences are tagged with the same pair of universal DNA segments. 23. The method of claim 16, wherein the first detectable signal is measured at every PCR cycle (real-time PCR). 24. The method of claim 16, wherein the first detectable signal is detected as an end point subsequent to PCR. 25. The method of claim 9, wherein the first detectable signal is detected as an end-point subsequent to PCR, and wherein the method further comprises counting the number of wells, nano-wells, droplets, DNA array features, or beads from which the detectable signal is emitted. 26. The method of claim 4, wherein the multiple target nucleic acids are selected from: a DNA, an RNA, a primate nucleic acid, a rodent nucleic acid, a viral nucleic acid, a bacterial nucleic acid, an archaea nucleic acid, a cDNA, a bisulphite treated DNA, a cDNA corresponding to a short RNA, a genetic variant, a mutation or insertion that confers drug resistance, a somatic mutation, a polymorphism, a single nucleotide polymorphism, a rare allele, a portion of the KRAS gene, a nucleic acid that exhibits a variation in copy number, an intron, an exon, an intron-exon boundary, a splice junction, one or more dinucleotides corresponding to one or more methylated or unmethylated CpG dinucleotides, a nucleic acid comprising one or more restriction enzyme recognition sequences, a portion of human chromosome 21, a portion of the human X chromosome, and a portion of the human Y chromosome, and wherein the presence and/or quantity of the multiple target nucleic acids is detected and combined into a diagnostic output. 27. The method of claim 26, wherein the diagnostic output is diagnostic for fetal aneuploidy, fetal sex, and/or fetal copy number variation, and wherein the fetal aneuploidy, fetal sex, and/or fetal copy number variation is detected by digitally counting nucleic acid targets indicative of fetal aneuploidy, fetal sex, and/or fetal copy number variation from a maternal blood sample. 28. The method of claim 27, wherein the nucleic acid targets indicative of fetal aneuploidy, fetal sex, and/or fetal copy number variation are detected using a first detectable label, and control nucleic acid targets are detected using a second detectable label, wherein the ratio of digital counts of the labels is used to detect fetal aneuploidy, fetal sex, and/or fetal copy number variation. 29. The method of claim 15, wherein the first universal primer is attached to a surface, wherein the label is disposed between the 5' and 3' portions of the first universal primer, wherein the label quencher or FRET dye is disposed at a 5' position relative to the label, wherein the first or second universal primer is extended by a polymerase wherein the 5' universal segment with label quencher or FRET dye hybridizes to the amplicon, and wherein extension of the universal primer removes the quencher or FRET dye from the surface-bound universal segment, thereby resulting or causing a change in the first detectable signal on the surface. 30. The method of claim 29, wherein the second universal primer is in solution or attached to the surface, and optionally the first universal primer comprises two oligos attached to the surface in sufficiently high density such that 3' and 5' solution ends of the two oligos are sufficiently spatially close to each other to anneal to the same first molecule. 31. The method of claim 8, wherein the one or more different reaction locations of step (b) are disposed within or upon a detection plate or fluidic device, and wherein the first and second universal primers are preloaded into the one or more different reaction locations. 32. The method of claim 4, wherein pools of encoding primers are inventoried or made to order, wherein the encoding primers are capable of amplifying a nucleic acid target selected from a gene, a portion of a gene, a single nucleotide polymorphism, a nucleic acid target that permits detection of a copy number variation, a methylation target, an miRNA, and a somatic mutation, and wherein a combination of one or more pools of the encoding primers is provided. 33. The method of claim 32, wherein the combination of one or more primer pools comprises a first pool of primers encoding a set of targets that optionally includes somatic mutations, single nucleotide polymorphisms, copy number variants, and/or methylation targets wherein the combination of one or more primer pools further comprises a second pool of encoding primers capable of amplifying one or more cDNAs or miRNAs to determine gene expression levels. 34. The method of claim 33, wherein both pools are used on the same biological sample, and wherein the results of both pools can be combined and analyzed together. 35. The method of claim 21, wherein the first and second universal primers comprise fluorescent labels having different emission wavelengths, wherein the first universal primer comprises a 5' portion that is substantially identical to a first strand of an internal portion of the tagged target nucleic acid, wherein the second universal primer comprises a 5' portion that is substantially identical to the complementary strand of the internal portion of the tagged target nucleic acid, wherein the 5' portion of the first universal primer and the 5' portion of the second universal primer are sufficiently similar to the first strand and the complementary strand of the tagged target nucleic acid, respectively, to anneal to the complement under PCR reaction conditions, and wherein amplification of both strands of the tagged target nucleic acid is measured independently using two or more colors. 36. The method of claim 35, wherein the target nucleic acid comprises two closely spaced polymorphisms or methylation sites, and wherein the two closely spaced polymorphisms or methylation sites are haplotyped using four colors.

Details for Patent 8,940,487

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2030-02-09
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2030-02-09
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2030-02-09