Nucleic acid amplification method

• US 7,790,388 B2
• Filed: 11/28/2007
• Issued: 09/07/2010
• Est. Priority Date: 08/03/2001
• Status: Expired

• Current Assignee: Olink AB
• Original Assignee: Olink AB
• Inventors: Nilsson, Mats, Landegren, Ulf, Gullberg, Mats
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Thomas; David C
• Application Number:
  US11/946,706
• Publication Number:
  US 20080131899A1
• Time in Patent Office: 1,014 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

A nucleic acid amplification method, and probes for use within the method are described.

Claims

1. A method of generating circularized nucleic acid, said method comprising:

providing a single-stranded target nucleic acid;

hybridizing the single-stranded target nucleic acid with a circularization adaptor having

(i) single-stranded sequences at each end and on the same strand, which single-stranded sequences are complementary to the ends of the single-stranded target nucleic acid and

(ii) a double-stranded non-target complementary segment between said single-stranded end sequences; and

circularizing the target nucleic acid using a DNA ligase.

2. The method of claim 1, wherein said circularization adaptor contains a restriction oligonucleotide sequence in said double-stranded segment.

3. The method of claim 1, further comprising amplifying said circularized nucleic acid.

4. The method of claim 3, wherein said amplification is rolling circle amplification.

5. The method of claim 1, wherein said single-stranded target nucleic acid is obtained by fragmenting a double-stranded nucleic acid using one or more restriction enzymes or FLAP endonucleases and then denaturing the resulting fragments.

6. The method of claim 1, wherein a multiplicity of different nucleic acid fragments are circularized in parallel using different circularization adaptors.

7. The method of claim 6, wherein the different circularization adaptors contain the same restriction oligonucleotide sequence and wherein a multiplicity of different nucleic acid fragments are amplified in parallel.

8. The method of claim 1, wherein the circularized nucleic acid comprises cDNA or genomic DNA.

9. A method of amplifying a target nucleic acid fragment, said method comprising:

(i) fragmenting a target nucleic acid,

(ii) circularizing a target nucleic acid fragment thus obtained, using a circularization adaptor having single-stranded sequences at each end complementary to the ends of the target fragment and, between said single-stranded end sequences, a double-stranded non-target-complementary segment, and

(iii) amplifying said circularized target nucleic acid fragment.

10. A method of analyzing at least one circularized nucleic acid, the method comprising amplifying the said circularized nucleic acid to provide an amplification product free in solution which comprises a concatemer of a sequence to be analysed coiled into a ball, the method further comprising the step of:

directly detecting by microscopic analysis said concatemer amplification product ball which is free in solution in a homogeneous detection reaction by hybridizing hybridization probes being fluorescence-labelled oligonucleotides to said amplification product ball, wherein the hybridization of the oligonucleotides results in an enrichment of the oligonucleotides in said amplification product ball and said enrichment causes a condensed signal which can be directly detected by microscopic analysis due to the contrast between said condensed signal and the diffuse signal from non-bound oligonucleotides.

11. The method of claim 10, wherein the signalling probes are singly or ratio-labelled.

12. The method claim 10, wherein the circularized nucleic acid to be analysed is a probe sequence.

13. The method of claim 10, wherein the circularized nucleic acid to be analysed comprises cDNA, genomic DNA or RNA sequences.

14. The method of claim 10, wherein the circularized nucleic acid to be analysed is circularized using cDNA, genomic DNA, or RNA sequence.

15. The method of claim 10, wherein said circularized nucleic acid is formed by hybridizing to a linear nucleic acid an oligonucleotide complementary to both end sequences of said linear nucleic acid and ligating said end sequences.

16. A method for generating a circularized nucleic acid molecule from two oligonucleotides, being parts of a pair of proximity probes, that have been joined in a proximity-dependent nucleic acid ligation reaction to form a joined product, said method comprising:

hybridizing a restriction oligonucleotide to two identical sequences present in both of the joined oligonucleotides in said joined product, digesting said joined product at the restriction sites thus created to release a linear nucleic acid molecule, and circularizing said released linear nucleic acid molecule by ligation by using an intact restriction oligonucleotide as a ligation template, said intact restriction oligonucleotide being complementary to both ends of said released linear molecule.

17. The method of claim 16, further comprising amplifying said circularized nucleic acid molecule.

18. The method of claim 17, wherein said amplification is rolling circle amplification.

19. The method of claim 16, wherein said restriction oligonucleotide is present in excess.

20. The method of claim 16, further comprising, after the digesting step, a heating step that inactivates the restriction enzyme and denatures the joined product.

21. The method claim 16, wherein the ligatable ends of the oligonucleotides that have been joined to form a joined product constitute unique half-tag sequences.

22. The method of claim 16, wherein the oligonucleotides that have been joined to form a joined product are joined by blunt-end ligation.