SEQUENCE BASED GENOTYPING BASED ON OLIGONUCLEOTIDE LIGATION ASSAYS

• US 20140221217A1
• Filed: 07/09/2012
• Published: 08/07/2014
• Est. Priority Date: 07/08/2011
• Status: Active Grant

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Van Eijk, Michael Josephus Theresia, Hogers, Rene Cornelis Josephus
• Granted Patent: US 9,777,322 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

Abstract

The invention relates to a method for the detection of a target nucleotide sequence in a sample based on an oligonucleotide ligation assay wherein probes are used that contain (a combination of) sequence-based identifiers that can identify the sample and the target sequence (i.e. locus and/or allele combination) wherein after the ligation step, the ligated probes, or after amplification, the amplified ligated probes, are restricted using restriction enzymes to cut of part of the probes and continue with those parts (identifiers and target sequence) that contain the relevant information in the sequencing step.

Claims

1. A method for the determination of a target nucleotide sequence in a sample comprising the steps of

(a) providing for each target nucleotide sequence (T) a first probe (P1) and a second probe (P2),

wherein the first probe comprises a first target specific section (TS1) and a first tag section (TAG1) that is non-complementary to the target nucleotide sequence and that optionally comprises a first primer binding sequence (PBS 1), wherein the first tag section comprise a first recognition sequence (RE1) for a first restriction endonuclease;

wherein the second probe comprises a second target specific section (TS2) and a second tag section (TAG2) that is non-complementary to the target nucleotide sequence and that comprises an optional second primer binding sequence (PB S2), wherein second tag section comprises an optional second recognition sequence (RE2) for a second restriction endonuclease;

(b) allowing the first and second target specific section of the respective first and second probe to hybridize to the target sequence;

(c) ligating the first and second probe when the respective target specific sections of the probes are hybridized to essentially adjacent sections on the target sequence to provide ligated probes (LP);

(d) optionally amplifying the ligated probes with an optional first and/or an optional second primer to provide amplicons (A);

(e) restricting the ligated probes or amplicons with the first and/or second restriction endonuclease to provide restricted ligated probes (RLP) or restricted amplicons (RA), ligating a first and/or a second adapter comprising an adapter-based identifier (AD ID1, AD ID2) to the restricted ligated probes (RLP) or restricted amplicons (RA).

(f) subjecting the adapter-ligated restricted ligated probes (RLP) or adapter-ligated restricted amplicons (RA) to high throughput sequencing technology to determine at least part of the nucleotide sequence of the restricted ligated probes or restricted amplicons;

(g) identifying the presence, absence or amount of the target nucleotide sequence in the sample.

2. The method according to claim 1, wherein the first target specific section is located at the 3′

-end of the first probe and wherein the second target specific section is located at the 5′

-end of the second probe.

3. The method according to claim 1, wherein a first probe-based identifier sequence (ID1) is located in the first tag section.

4. The method according to claim 1, wherein a second probe-based identifier sequence (ID2) is located in the second tag section.

5. The method according to claim 1, wherein the first probe-based identifier is located between the first recognition sequence for a restriction endonuclease and the first target specific section.

6. The method according to claim 1, wherein the second probe-based identifier is located between the optional second recognition sequence for a restriction endonuclease and the second target specific section.

7. The method according to claim 1, wherein the first and the second tag section both contain comprise a recognition site for a restriction endonuclease.

8. The method according to claim 1, wherein the hybridization, ligation and optional gap filling are performed in a combined step.

9. The method according to claim 1, wherein the recognition sequence for the restriction endonuclease for the first tag section has a different nucleotide sequence compared to the recognition site for the restriction endonuclease for the second tag section.

10. The method according to claim 1, wherein the high throughput sequencing comprises sequencing by synthesis.

11. The method according to claim 1, wherein the high throughput sequencing comprises bridge amplification or emulsion amplification.

12. The method according to claim 1, wherein the high throughput sequencing comprises unidirectional single read sequencing.

13. The method according to claim 1, wherein the high throughput sequencing unidirectional single read double priming sequencing.

14. The method according to claim 1, wherein the high throughput sequencing comprises bidirectional (paired end) sequencing.

15. The method according to claim 1, high throughput sequencing comprises mate pair sequencing.

16. The method according to claim 1, wherein a plurality of target sequences is determined in one sample.

17. The method according to claim 1, wherein one target sequence is determined in a plurality of samples.

18. The method according to claim 1, wherein a plurality of target sequences is determined in a plurality of samples.

19. The method according to claim 1, wherein the probes are circularizable probes, keylock probes and/or compound probes.

20. A method for genotyping a biological sample for the presence, absence or amount of a target sequence in the sample using an oligonucleotide ligation assay comprising at least two probes wherein at least one of the probes comprises a recognition sequence for a restriction endonuclease.

21. The method according to claim 20, wherein the method further comprises a ligation step to provide ligated probes.

22. The method according to claim 21, wherein the method further comprises an amplification step to provide amplicons.

23. The method according to claim 21, wherein the method further comprises a step wherein the amplicons or ligated probes are contacted with a restriction endonuclease to provide restricted amplicons and/or restricted ligated probes.

24. The method according to claim 20, wherein the genotyping is co-dominant genotyping comprising at least two allele-specific probes.

25. The method according to claim claim 20, wherein at least part of the sequence of part of the restricted amplicons or restricted ligated probes is determined.