Nucleic acid encoding reactions

Patent metadata

Field	Value
Publication number	US-12018323-B2
Application number	US-202117480067-A
Country	US
Kind code	B2
Filing date	Sep 20, 2021
Priority date	May 20, 2011
Publication date	Jun 25, 2024
Grant date	Jun 25, 2024

Abstract

Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.

First claim

What is claimed is:

1. A method for detecting, and/or quantifying the relative amounts of, at least two different target nucleic acids in a nucleic acid sample, the method comprising:
conducting reactions in a single reaction mixture to produce producing first and second tagged target nucleotide sequences from first and second target nucleic acids in the sample,
the first tagged target nucleotide sequence comprising a first nucleotide tag; and
the second tagged target nucleotide sequence comprising a second nucleotide tag, wherein the first and second nucleotide tags are different, allele-specific tags,

wherein 1-3 reactions are performed in the single reaction mixture;
subjecting the tagged target nucleotide sequences to:
a first primer extension reaction using a first primer that anneals to the first nucleotide tag; and
a second primer extension reaction using a second primer that anneals to the second nucleotide tag; and

detecting and/or quantifying:
a signal that indicates extension of the first primer; and
a signal that indicates extension of the second primer
wherein the signal for a given primer indicates the presence, and/or relative amount of, the corresponding target nucleic acid.

2. The method of claim 1 , wherein the first and second tagged target nucleotide sequences comprise adaptors for DNA sequencing at the ends of each molecule.

3. The method of claim 1 , wherein the first and second tagged target nucleotide sequences are produced by amplifying first and second target nucleic acids with first and second primer pairs, respectively, wherein at least one primer in the first primer pair comprises a first nucleotide tag and at least one primer in the second primer pair comprises a second nucleotide tag.

4. The method of claim 3 , wherein one primer in each primer pair comprises 5′-(DNA sequencing adaptor)-(nucleotide tag)-(target-specific portion)-3′ and the other primer in each primer pair comprises 5′-(DNA sequencing adaptor)-(target-specific portion)-3′.

5. The method of claim 3 , wherein the tagged target nucleotide sequences are further amplified prior to primer extension.

6. The method of claim 5 , wherein said further amplification comprises emulsion amplification or bridge amplification.

7. The method of claim 1 , wherein the first and second primer extension reactions are carried out sequentially in at least two cycles of primer extension, wherein:
a first cycle of primer extension is carried out using the first primer that anneals to the first nucleotide tag;
a second cycle of primer extension is carried out using the second primer that anneals to the second nucleotide tag;
all deoxynucleoside triphosphates are provided in each cycle of primer extension;
the incorporation of any deoxynucleoside triphosphate into a DNA molecule produces a detectable signal; and
the signal detected in the first cycle indicates the presence, and/or relative amount, of the first target nucleic acid in the nucleic acid sample, and
the signal detected in the second cycle indicates the presence, and/or relative amount, of the second target nucleic acid in the nucleic acid sample.

8. The method of claim 1 , wherein the detectable signal comprises pyrophosphate release.

9. The method of claim 7 , wherein the tagged target nucleotide sequences are further amplified by emulsion PCR prior to primer extension.

10. The method of claim 1 , wherein the first and second primer extension reactions are carried out by oligonucleotide ligation and detection, and wherein:
the ligation of a labeled di-base oligonucleotide to the first and/or second primer(s) produces a detectable signal; and
the total signal detected for a particular primer indicates the presence, and/or relative amount, of the corresponding target nucleic acid in the nucleic acid sample.

11. The method of claim 10 , wherein the ligation of a labeled di-base oligonucleotide to the first primer produces the same detectable signal as the ligation of a labeled di-base oligonucleotide to the second primer, and the first and second primer extension reactions are carried out separately.

12. The method of claim 10 , wherein the ligation of a labeled di-base oligonucleotide to the first primer produces a different detectable signal than the ligation of a labeled di-base oligonucleotide to the second primer.

13. The method of claim 12 , wherein the first and second primer extension reactions are carried out simultaneously, in one reaction mixture.

14. The method of any of claim 10 , wherein the tagged target nucleotide sequences are further amplified by emulsion PCR prior to primer extension.

15. The method of claim 1 , wherein the first and second primer extension reactions comprise sequencing-by-synthesis, wherein:
each deoxynucleoside triphosphates is labeled with a distinct, base-specific label;
the incorporation of a deoxynucleoside triphosphate into a DNA molecule produces a base-specific detectable signal; and
the total signal detected for a particular primer indicates the presence, and/or relative amount, of the corresponding target nucleic acid in the nucleic acid sample.

16. The method of claim 15 , wherein the extension of the first primer produces the same detectable signal as the extension of the second primer, and the first and second primer extension reactions are carried out separately.

17. The method of claim 15 , wherein the extension of the first primer produces a different detectable signal than the extension of the second primer.

18. The method of claim 15 , wherein the tagged target nucleotide sequences are further amplified by bridge PCR prior to primer extension.

19. The method of claim 5 , wherein amplification produces clonal populations of tagged target nucleotide sequences that are, or become, located at discrete reaction sites.

20. The method of claim 19 , wherein the number of reaction sites comprising the first nucleotide tag relative to the number of reaction sites comprising the second nucleotide tag indicates the amount of the first target nucleic acid relative to the second target nucleic acid in the sample.

21. The method of claim 19 , wherein said detecting and/or quantifying comprises:
detecting and comparing the total signal from all reaction sites comprising the first nucleotide tag with the total signal from all reaction sites comprising the second nucleotide tag; or
detecting and comparing the number of reaction sites comprising the first nucleotide tag with the number of reaction sites comprising the second nucleotide tag.

22. The method of claim 1 , wherein the first nucleotide tag comprises a poly-(first nucleotide) sequence, and the second nucleotide tag comprises a poly-(second nucleotide) sequence, wherein the first and second nucleotides are different.

23. The method of claim 1 , wherein the first and second target nucleic acids are selected from:
two different alleles of a polymorphic site;
a target nucleic acid that may be present in the nucleic acid sample at an altered copy number and a reference target nucleic acid that is expected to be present in the sample at a normal copy number;
target nucleic acids on a single chromosome; and
target nucleic acids on different chromosomes.

24. The method of claim 1 , the method comprising:
producing three or more tagged target nucleotide sequences from three or more target nucleic acids in the sample
the tagged target nucleotide sequences are subjected to three o

Assignees

• Fluidigm Corp

Inventors

• Anderson Megan
• Chen Peilin
• Fowler Brian
• Jones Robert C
• Kaper Fiona
• Lebofsky Ronald
• May Andrew

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title