Rolling circle amplification assay for nucleic acid analysis

• US 6,221,603 B1
• Filed: 02/04/2000
• Issued: 04/24/2001
• Est. Priority Date: 02/04/2000
• Status: Expired due to Fees

• Current Assignee: GE Healthcare SV Corp (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Dynamics Inc. (General Electric Company)
• Inventors: Mahtani, Melanie M.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/498,585
• Time in Patent Office: 445 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

Abstract

Method and reagents for analysis of nucleic acid sequences is disclosed. In this method a plurality of padlock probes is provided. Each padlock probe may hybridize to a locus on a target nucleic acid under hybridization conditions. If a targeted variant is present at the locus, the padlock probe may be ligated to form an amplification target circle. The amplification target circle acts as a template for production of tandem-sequence DNA. The tandem-sequence DNA may then be digested into non-tandem detection fragments which are subsequently separated and detected. The plurality of padlock probes are designed such that ligation of the probes, amplification of the target circle, and digestion of the tandem-sequence DNA subsequently produced, and detection may all be effected with the same set of reagents. Each probe targets a unique locus variant on the target nucleic acid sequence and produces a detection fragment that may be distinguished from detection fragments produced from other padlock probe in the plurality of padlock probes by using a fragment analysis detector.

Claims

1. A plurality of padlock probes, each probe comprising:

a nucleic acid sequence, the sequence including two target hybridization end regions on opposing ends of each padlock probe, the target hybridization end regions complementary to a target hybridization region on a single strand target nucleic acid sequence, wherein the target hybridization end regions may hybridize to the single strand target nucleic acid sequence to form a probe/target duplex, wherein each pair of target hybridization end regions are proximate to each other when hybridized to the target nucleic acid strand; and

a stuffer region located between said target hybridization end regions, said stuffer region having a primer complementary sequence and a restriction site complementary sequence, wherein each of said padlock probes has the same primer complementary sequence and restriction site complementary sequence;

wherein each probe may act as a template for production of tandem sequence DNA by ligation and amplification of the probe if a specific target nucleic sequence is present;

wherein tandem sequence DNA produced from each template padlock probe may be segmented into a set of non-tandem detection fragments of a discrete length, wherein the discrete length of detection fragments produced from each padlock probe differs in length from any detection fragment produced from any other padlock probe in the plurality of padlock probes.

2. The plurality of padlock probes of claim 1, wherein the target hybridization end regions of each padlock probe are complementary to a target hybridization region on the single strand nucleic acid such that there is a gap between said two target hybridization end regions.

3. The plurality of padlock probes of claim 2, wherein each gap is at least 4 bases in length.

4. The plurality of padlock probes of claim 2, wherein the locus on the target nucleic acid opposite the gap is a locus of a single nucleotide polymorphism.

5. The plurality of padlock probes of claim 1, wherein each target hybridization end region is at least 10 bases long.

6. The plurality of padlock probes of claim 1 wherein each stuffer region includes a constant region and a variable region, wherein the constant region is identical for each of the padlock probes of the plurality of padlock probes and wherein the variable region is non-identical for each of the probes in the plurality of padlock probes, wherein the constant region contains the primer complementary sequence, and wherein the variable region is of a different length for each of padlock probes in the plurality of padlock probes.

7. The plurality of padlock probes of claim 6 further comprising two restriction site complementary sequences in said constant region of the stuffer region, each restriction site complementary sequence located proximate to one of the two target hybridization end regions and each of said two restriction site complementary sequences identical for each of the plurality of padlock probes.

8. The plurality of padlock probes of claim 7, wherein the two restriction enzyme site complementary sequences are the same.

9. The plurality of padlock probes of claim 1 wherein the discrete length of detection fragments produced from each padlock probe differs in length from any other detection fragment produced from any other padlock probe in the plurality of padlock probes by at least two bases.

10. The plurality of padlock probes of claim 6 wherein the restriction enzyme site template is a single strand restriction enzyme site template.

11. The plurality of padlock probes of claim 6 wherein the restriction site template is for a restriction enzyme that cuts double-stranded DNA and wherein the restriction site template is located within the primer complementary sequence.

12. A method for high throughput analysis of genetic loci, the method comprising the steps:

a) providing a target nucleic acid sample;

b) combining the target nucleic acid sample with a plurality of padlock probes to form padlock probe/target hybrids, wherein each probe is a nucleic acid sequence including,

two target hybridization end regions on opposing ends of the padlock probe, the target hybridization end regions complementary to a target hybridization region on a single strand target nucleic acid sequence, wherein the target hybridization end regions hybridize to the single strand target nucleic acid sequence to form a probe/target duplex, wherein each pair of target hybridization end regions are proximate to each other when hybridized to the target nucleic acid strand, and

a stuffer region located between said target hybridization end regions, said stuffer region having a primer complementary sequence and restriction site complementary sequence, wherein each of said plurality of said padlock probes has the same primer complementary sequence and restriction site complementary sequence;

c) mixing ligase with the padlock probe/target nucleic acid duplex to form a ligation mixture, and incubating the ligation mixture under conditions that promote ligation of the padlock probes to form amplification target circles;

d) mixing a primer which is complementary to said primer complementary sequence with the amplification target circles, and incubating the primer/amplification target circles mixture under conditions that promote hybridization between the amplification target circles and the primers;

e) mixing a DNA polymerase with the primer/amplification target circles mixture, to produce a rolling circle replication mixture, and incubating the rolling circle amplification mixture under conditions that promote rolling circle replication of the amplification target circles,

wherein replication of the amplification target circles results in the formation of tandem sequence DNA;

f) cutting each tandem sequence DNA at the generated restriction enzyme site into sets of non-tandem DNA fragments of a discrete length, wherein each amplification target circle produces tandem sequence DNA that is cut into sets of non-tandem DNA fragments of a unique discrete size;

g) separating by length said sets of non-tandem DNA fragments; and

h) detecting said sets of separated non-tandem DNA fragments.

13. The method of claim 12, wherein steps b, c, d, e and f all are carried out in a single reaction container.

14. The method of claim 12 wherein the step of combining the target nucleic acid sample with a plurality of padlock probes includes combining the target nucleic acid with 30 padlock probes.

15. The method of claim 13 wherein each padlock probe will be ligated to form an amplification target circle only if a specific polymorphism at a genomic loci is present on the target nucleic acid sequence.

16. The method of claim 13 wherein each padlock probe will be ligated to form an amplification target circle only if a specific sequence at a specific locus on the target nucleic acid is present.

17. The method of claim 13 wherein each padlock probe will be ligated to form an amplification target circle only in the presence of a target mRNA molecule.

18. The method of claim 13 wherein each padlock probe will be ligated to form an amplification target circle only in the presence of a target cDNA molecule.

19. The method of claim 12 wherein the padlock probe includes a labeled secondary probe complement region on each of the plurality of padlock probes whereby step h may be effected by adding a labeled secondary probe that hybridizes to the secondary probe complement region and detecting the label on the secondary probe whereby each set of fragments may be detected by detecting the same secondary probe label.

20. The method of claim 19, wherein the label is a fluorescent dye.

21. The method of claim 12 wherein step g is effected by capillary electrophoretic separation.

22. The method of claim 21 further including prior to step g the step of adding a sizing ladder to the sets of non-tandem DNA fragments.

23. The method of claim 12, wherein each of the non-tandem DNA fragments of each set of non-tandem DNA fragments differs in length from any other non-tandem DNA fragment of any other set of non-tandem DNA fragments by at least 2 bases.

24. The method of claim 23 wherein non-tandem DNA fragments produced are between 20 to 150 bases in length.

25. The method of claim 12 wherein the step of cutting the tandem sequence DNA at the generated restriction site includes cutting the tandem sequence DNA at a single stranded restriction enzyme cutting site.

26. The method of claim 20 wherein the step of cutting each tandem sequence into sets of non-tandem fragments is effected by providing a plurality of padlock probes wherein each padlock probe has a first and a second restriction enzyme template each located proximate to one of the two target hybridization end regions such that tandem sequence DNA produced by each of the plurality of padlock probes may be cut into two sets of fragments, a first set of fragments complementary to the two target hybridization end regions and a second set of fragments wherein the second set of fragments generated from any tandem sequence DNA is of different length for each tandem sequence DNA generated from each unique amplification target circle.

27. The method of claim 26 wherein the first and second restriction enzyme site templates are the same restriction enzyme template.

28. The method of claim 12 wherein the method detects variation in single nucleotide polymorphisms.