Methods and compositions for universal detection of nucleic acids

• US 9,938,570 B2
• Filed: 12/10/2014
• Issued: 04/10/2018
• Est. Priority Date: 02/09/2010
• Status: Active Grant

• Current Assignee: Unitaq Bio
• Original Assignee: Unitaq Bio
• Inventors: Spier, Eugene
• Primary Examiner(s): TREPTOW, NANCY ANN
• Application Number:
  US14/566,596
• Publication Number:
  US 20150167067A1
• Time in Patent Office: 1,217 Days
• Field of Search: None
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

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.

Claims

1. An analyte nucleic acid detection reaction mixture, used to detect a target nucleic acid subsequence of interest, the mixture comprising:

an analyte nucleic acid comprising the target nucleic acid subsequence of interest, the analyte nucleic acid further comprising first, second and third tag sequences, the second tag sequence being located between the first and third tag sequences; and

both the first and the second tag sequences are universal tag sequences;

a first universal primer comprising a first tag complement subsequence that is complementary to the first tag sequence, a subsequence that comprises the second tag sequence, and a detectable label; and

,

a second universal primer comprising a sequence that comprises the third tag sequence.

2. The reaction mixture of claim 1, wherein the mixture further comprises a complementary nucleic acid comprising subsequences complementary to the nucleic acid subsequence of interest and the first, second and third tag sequences.

3. The reaction mixture of claim 1, wherein the nucleic acid subsequence of interest comprises a nucleic acid or nucleic acid feature 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 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.

4. The reaction mixture of claim 1, wherein the first universal primer comprises a label quencher disposed at an effective quenching distance from the label.

5. The reaction mixture of claim 4, wherein the label quencher is located between the first tag complement subsequence and the subsequence that comprises the second tag sequence, and wherein the label is located on an end opposite the subsequence that comprises the second tag sequence, as compared to the label quencher.

6. The reaction mixture of claim 4, wherein the label is located between the first tag complement sequence and the subsequence that comprises the second tag sequence, and wherein the label quencher is located on an end opposite the subsequence that comprises the second tag sequence, as compared to the label.

7. The reaction mixture of claim 4, wherein the first universal primer comprises a polymerase blocking unit between the first tag complement and second tag sequence.

8. The reaction mixture of claim 1, wherein the label is a fluorescent label.

9. The reaction mixture of claim 1, wherein first universal primer is configured to generate a detectable signal when the 3′

end of the first universal primer is extended.

10. The reaction mixture of claim 1, further comprising:

a second analyte nucleic acid comprising a second target nucleic acid subsequence of interest, the second analyte nucleic acid further comprising fourth, fifth and sixth tag sequences, the fifth tag sequence being located between the fourth and sixth tag sequences;

a third universal primer comprising a second tag complement subsequence that is complementary to the fourth tag sequence, a subsequence that comprises the fifth tag sequence, a label and a label quencher, wherein the label of the second universal label primer is different from the label of the first universal label primer; and

,

a fourth universal primer comprising a sequence that comprises the sixth tag sequence.

11. The reaction mixture of claim 10, wherein the sixth tag sequence and the third tag sequence are the same.

12. The reaction mixture of claim 10, wherein the second nucleic acid subsequence of interest comprises a nucleic acid or nucleic acid feature selected from:

a DNA, an RNA, a bisulphite treated DNA, 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.

13. The reaction mixture of claim 10, wherein the first nucleic acid subsequence of interest comprises a cDNA sequence corresponding to an mRNA expressed from a first gene, and wherein the second nucleic acid subsequence of interest comprises a cDNA sequence corresponding to an mRNA expressed from a second gene.

14. The reaction mixture of claim 1, wherein the first universal primer comprises two oligonucleotides attached to a surface at a density such that 3′

and 5′

solution ends of the two oligonucleotides are spatially close to each other so as to function as two ends of the first universal primer.

15. The reaction mixture of claim 1, wherein the first universal primer comprises the second tag sequence and a complement of the first tag sequence and, and the second universal primer comprises the third tag sequence.

16. The reaction mixture of claim 1, wherein the nucleic acid sequence of interest does not comprise the second tag sequence.

17. The reaction mixture of claim 1, further comprising:

a second analyte nucleic acid comprising a second nucleic acid sequence of interest, the second analyte nucleic acid further comprising fourth, fifth, and sixth tag sequences, the fifth tag sequence being located between the fourth and sixth tag sequences;

a third universal primer comprising a fourth tag complement sequence, the fifth tag sequence, a label and a label quencher, wherein the label of the second universal primer is the same as the label of the first universal primer; and

,

a fourth universal primer comprising a sequence that comprises the sixth tag sequence.

18. The reaction mixture of claim 17, wherein the fourth tag sequence is the same as the first tag sequence of the first analyte nucleic acid.

19. The reaction mixture of claim 1, wherein the analyte nucleic acid is a construct wherein the target nucleic acid subsequence of interest is a natural genomic sequence, and the second tag sequence is not a sequence naturally found adjacent to the subsequence of interest.

20. The reaction mixture of claim 19, wherein the second or third tag sequence was previously attached to the sequence of interest in a PCR extension reaction.

21. The reaction mixture of claim 19, wherein the first or second tag sequence was not previously attached to the sequence of interest before a polymerase extension of the target sequence of interest.

22. The reaction mixture of claim 1, wherein the mixture has not been exposed to a polymerase extension reaction or does not comprise a DNA polymerase.

23. The reaction mixture of claim 1, wherein the analyte nucleic acid has not been transcribed by extension from the first universal primer.

24. The reaction mixture of claim 1, wherein the analyte nucleic acid comprises sequences in the order of:

first tag sequence, target nucleic acid of interest sequence, second tag sequence.

25. The reaction mixture of claim 1, wherein the mixture is configured so that extension of the first universal primer along the analyte nucleic acid provides an extension product comprising sequences in the order of:

complement of the first tag sequence, complement of the nucleic acid of interest sequence, and complement of the second tag sequence.

26. The reaction mixture of claim 25, wherein extension of the first universal primer along the analyte nucleic acid provides an extension product comprising the features in the order of:

the detectable label, the second tag sequence, complement of the first tag sequence, complement of the nucleic acid of interest sequence, complement of the second tag sequence.

27. The reaction mixture of claim 1, wherein extension of the first universal primer along the analyte nucleic acid provides an extension product comprising the features in the 3′

to 5′

order of;

the second tag sequence, complement of the first tag sequence, complement of the nucleic acid of interest sequence, complement of the second tag sequence.

28. The reaction mixture of claim 1, wherein the first tag sequence is a universal tag sequence complementary to the first universal primer and third tag sequences is a universal tag sequences complementary to the second universal primer.

29. The reaction mixture of claim 1, wherein the universal tag sequences are not related to the nucleic acid of interest.