Method of detecting target nucleic acid molecules

A method of decoding a nucleotide barcode sequence in a nucleic acid molecule, wherein the nucleotide barcode sequence corresponds to a specific signal code sequence that may be derived by interrogating the nucleotide barcode sequence with sequential detection probes each yielding a signal and the signals together make up the signal code sequence, and wherein the nucleotide barcode sequence comprises at least two adjacent barcode positions, each barcode position comprising a barcode subunit pair comprising a first barcode subunit and a second barcode subunit, wherein the second barcode subunit from each barcode position at least partially overlaps with the first barcode subunit of the adjacent barcode position in the sequence, said method comprising:

• (i) contacting the nucleic acid molecule with a first detection probe for generating a signal code for the first position of the signal code sequence, and allowing the detection probe to hybridise to the nucleic acid molecule, wherein the detection probe comprises a recognition sequence that is complementary to the barcode subunit pair that is present at the first barcode position and a reporter;
• (ii) detecting a signal from the reporter of the first detection probe which has hybridized to the nucleotide barcode sequence at the first barcode position;
• (iii) identifying the signal code for the first position of the signal code sequence from the signal detected in (ii);
• (iv) contacting the nucleotide barcode sequence with a subsequent detection probe for generating a signal code for the next position of the signal code sequence, and allowing the detection probe to hybridise to the nucleic acid molecule, wherein the subsequent detection probe comprises a recognition sequence that is complementary to the barcode subunit pair that is present at the adjacent barcode position, and a reporter, and wherein the subsequent detection probe which hybridises to the barcode subunit at the adjacent position hybridises to a sequence within the overlapping portion of the preceding barcode position, and thereby initiates a strand displacement reaction which displaces the hybridised preceding detection probe;
• (v) detecting a signal from the reporter of the subsequent detection probe which has hybridized to the nucleotide barcode sequence at the adjacent barcode position;
• (vi) identifying the signal code for the next position of the signal code sequence from the signal detected in (v);
• (vii) repeating steps (iv)-(vi) to identify signal codes for subsequent signal code sequence positions until sufficient signal codes have been identified to decode the nucleotide barcode sequence, wherein detection probes for identifying the subsequent signal codes in the signal code sequence have (a) recognition sequences that are complementary to barcode subunit pairs that are present at subsequent sequential barcode positions in a nucleotide barcode sequence comprising multiple sequential adjacent barcode positions, or (b) recognition sequences that are complementary to the barcode subunit pairs that are present at the first and second barcode positions of a nucleotide barcode sequence comprising two adjacent barcode positions, and a reporter;
• wherein the first barcode position can be any barcode position in the nucleotide barcode sequence.

A method of detecting a target nucleic acid molecule, wherein the target nucleic acid molecule is detected using a padlock probe specific for said target molecule which is circularised upon hybridisation to a target sequence in the target nucleic acid molecule and which is provided with a nucleotide barcode sequence to identify the padlock probe, and wherein the circularised padlock probe is amplified by rolling circle amplification (RCA) to produce a rolling circle product (RCP), the RCP containing multiple complementary copies of the nucleotide barcode sequence, characterised in that the method comprises:

• (a) assigning a unique signal code sequence to the target nucleic acid molecule,
• wherein the signal code sequence is specific to that molecule;
• (b) providing the padlock probe with a nucleotide barcode sequence such that a RCP is formed comprising multiple complementary copies of the nucleotide barcode sequence that each corresponds to the signal code sequence, wherein the signal code sequence may be derived by interrogating the complementary copies of the nucleotide barcode sequence with sequential detection probes each yielding a signal and the signals together make up the signal code sequence;
• (c) contacting the RCP with a first detection probe for generating a signal code for the first position of the signal code sequence and allowing the detection probe to hybridise to the RCP at the complementary copies of the nucleotide barcode sequence, wherein the detection probe comprises a recognition sequence complementary to the nucleotide barcode sequence and a reporter;
• (d) detecting a signal from the reporter of the first detection probe which has hybridized to the RCP;
• (e) identifying the first signal code for the first position in the signal code sequence for the RCP from the signal detected in (d);
• (f) removing the first detection probe from the RCP;
• (g) contacting the RCP with a subsequent detection probe for generating a signal code for the next position of the signal code sequence, and allowing the detection probe to hybridise to the nucleic acid molecule at the complementary copies of the nucleotide barcode sequence, wherein the detection probe comprises a recognition sequence complementary to the nucleotide barcode sequence and a reporter;
• (h) detecting a signal from the reporter of the subsequent detection probe which has hybridized to the RCP;
• (i) identifying the signal code for the next position in the signal code sequence for the RCP from the signal detected in (g);
• (j) removing the subsequent detection probe from the RCP;
• (k) repeating steps (g)-(j) to identify signal codes for subsequent signal code positions until sufficient signal codes have been identified to identify the nucleotide barcode sequence and thereby to detect the nucleic acid molecule.