Patent ID: 11970736
Assignee: GENOMILL HEALTH OY
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method for the high-throughput detection of one or more target nucleotide sequences in a plurality of samples, the method comprising the steps of:
(i) providing for each target nucleotide sequence in each of the samples:
a first probe, a second probe, a barcode loop oligo and a bridge oligo or a plurality of bridge oligonucleotides capable of annealing to the barcode loop oligo to form a bridge oligo complex,
wherein the first probe comprises a first bridge oligo-specific sequence at the 5′ end of the first probe and a first target-specific portion at the 3′ end of the first probe;
wherein the second probe comprises a second target-specific portion at the 5′ end of the second probe and a second bridge oligo-specific sequence at the 3′ end of the second probe;
wherein the barcode loop oligo comprises, starting from the 5′ end of the molecule, a third bridge oligo-specific sequence, a barcoded loop sequence and a fourth bridge oligo-specific sequence,
wherein the bridge oligo or plurality of bridge oligonucleotides contains sequences complementary to the first bridge oligo-specific sequence and the second bridge oligo-specific sequence in the first probe and the second probe, respectively, and sequences complementary to the third bridge oligo-specific sequence and the fourth bridge oligo-specific sequence in the barcode loop oligo;
and wherein, optionally, at least one of: the first probe, the second probe, the barcode loop oligo, the bridge oligo or an oligonucleotide of the plurality of bridge oligonucleotides comprises a recognition sequence for an endonuclease;
(ii) contacting, for each of the one or more target nucleotide sequences, the first probe and the second probe with, for each of the samples in a separate tube, the barcode loop oligo and the bridge oligo or plurality of bridge oligonucleotides, and allowing self-annealing into ligation complexes, and wherein the third and fourth bridge oligo-specific sequences of the barcode loop oligo hybridize to a sequence of the bridge oligo or plurality of bridge oligonucleotides located between where the first and second bridge oligo-specific sequences of the respective first and second probes hybridize to the bridge oligo or plurality of bridge oligonucleotides;
(iii) contacting nucleic acids present in each of the samples to be tested for the target nucleotide sequences with the ligation complexes;
(iv) allowing the first target-specific portion and the second target-specific portion of the respective first probe and the second probe to hybridize to essentially adjacent sections on the target sequence, thereby forming hybridization complexes;
(v) optionally pooling the hybridization complexes from the plurality of samples;
(vi) ligating the probes in the hybridization complexes to provide ligated ligation complexes;
(vii) amplifying nucleic acids from the one or more ligated ligation complexes using rolling circle amplification with a strand-displacing polymerase thereby obtaining single-stranded concatemeric sequences;
(viii) optionally, provided a recognition sequence as specified in step (i) is present, performing a step to obtain nucleic acid fragments by:
(a) cleaving the single-stranded concatemeric sequences obtained in step (vii), or
(b) subjecting the single-stranded concatemeric sequences obtained in step (vii) to annealing with a specific oligonucleotide containing a recognition sequence for an endonuclease wherein the oligonucleotide anneals with the recognition sequence specified in step (i) such that a recognition site for the endonuclease is obtained and cleaving the annealed complexes with said endonuclease;

(ix) subjecting the concatemeric sequence obtained in step (vii) or the nucleic acid fragments obtained in step (viii) to high-throughput sequencing technology to determine the barcode sequence(s); and
(x) identifying the presence and/or number of the target nucleotide sequence in the plurality of samples by determination of at least part of the first target-specific portion and/or the second target-specific portion, and/or at least part of the barcode sequence corresponding to the barcode in the barcode loop oligo,
wherein steps (v) and (vi) may be performed in any order.