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OwnerMats NILSSON BERNITZ, Marco GRILLO +1inventors
InventorsMats NILSSON BERNITZ, Marco GRILLO, Hao Zhe LEE
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The disclosure relates to a method for profiling of mutations, insertions, deletions and/or single or nucleotide variations in a RNA sample via gap filling, comprising the steps of: (a) providing an RNA sample comprising at least one nucleotide stretch of unknown identity to be identified, flanked by nucleotide stretches of known identity; (b) contacting the RNA sample with at least one DNA probe, such as at least two linear probes or at least one DNA padlock probe having a first and a second end under conditions and with reagents allowing hybridization, which first and second ends are designed to hybridize to the sequences of known identity flanking the at least one nucleotide stretch of unknown identity, thereby resulting in a gap between the hybridized first and second ends of the at least one DNA probe; (c) adding a polymerase with reverse transcriptase activity, and optionally nick translation activity, and optionally limited strand displacement, under conditions and with reagents allowing reverse transcription, thereby allowing reverse transcription of the at least one DNA probe from the first to the second end using the RNA sample as a template, so that the reverse transcribed part of the at least one DNA probe comprises the complementary sequence to the nucleotide stretch of unknown identity in the RNA sample; (d) adding a ligase that can use RNA as a splint molecule under conditions and with reagents allowing ligation, thereby allowing sealing of the at least one reverse transcribed DNA probe; (e) the probe containing the reverse transcribed nucleotide(s) can then be amplified; and (f) sequencing the at least one amplified circularized oligonucleotide, thereby revealing the identity of the at least one nucleotide stretch of unknown identity of the RNA sample. The disclosure further relates to a kit for use in a method for the profiling of mutations for both in situ and in vitro applications.
A method for profiling of mutations, insertions, deletions and/or single or nucleotide variations in an RNA sample, comprising the steps of:
The method according to any one of the preceding claims, wherein the RNA sample is an mRNA, rRNA or other non-coding RNA sample.
The method according to any one of the preceding claims, wherein the at least one DNA probe is chosen from (i) at least one padlock probe, (ii) at least two linear probes that can be circularized by introducing a bridging probe and a ligase, (iii) at least one padlock probe or at least two linear probes that can be enzymatically ligated and can be released from the RNA sample, amplified and sequenced via NGS (next generation sequencing), or (iv) at least one padlock probe or at least two linear probes that can be chemically ligated via click reaction, thereby not being circularized and can be released from the RNA sample, amplified and sequenced via NGS.
The method according to any one of the preceding claims, wherein the first end of the at least one DNA probe is designed to hybridize to the RNA sample to the nucleotide stretch of known identity at the 3′ side of the nucleotide stretch of unknown identity, and the second end of the at least one DNA probe is designed to hybridize to the RNA sample to the nucleotide stretch of known identity at the 5′ side of the nucleotide stretch of unknown identity.
The method according to any one of the preceding claims, wherein the at least one DNA probe is either phosphorylated or unphosphorylated at the 5′ end.
The method according to any one of the preceding claims, wherein the lengths of the first and second ends of the at least one DNA probe that are designed to hybridize to the RNA sample to the nucleotide stretches of known identity are in the interval of 10-30 nucleotides.
The method according to any one of the preceding claims, wherein the gap between the first and second ends of the at least one DNA probe hybridized to the RNA sample, corresponding in length to the stretch of unknown identity to be identified, is in the interval of 0-20 nucleotides.
The method according to any one of the preceding claims, wherein the polymerase with reverse transcriptase activity is chosen from the group comprising BST DNA polymerase (Full Length), TTH DNA Polymerase and DNA Polymerase I, as well as engineered mutant polymerases derived from the thermophilic bacterium Bacillus stearothermophilus, family A of DNA polymerases and other polymerases and reverse transcriptases exhibiting low strand displacement activity and/or good RT fidelity.
The method according to any one of the preceding claims, wherein the polymerase with reverse transcriptase activity is a DNA polymerase with limited reverse transcription activity and nick translation activity that is derived from the thermophilic bacterium Bacillus stearothermophilus or family A of DNA polymerases.
The method according to any one of the preceding claims, wherein the ligase is chosen from Splint R ligase, PBCV-1 DNA ligase and/or Chlorella virus DNA ligase.
The method according to any one of the preceding claims, wherein the hybridized 5′ end of the DNA probe(s) in step (b) contains a 5′-alkyne group and after reverse transcription with nucleotides with a 3′-azide group, the nick between adjacent 5′-alkyne and 3′-azide group is to be chemically ligated via CuAAC click chemistry.
The method according to any one of the preceding claims, wherein steps (c) and (d) are combined, thereby allowing reverse transcription and sealing of the at least one DNA probe within the same step.
The method according to any one of the preceding claims, wherein an additional step of nick translation is performed together with the reverse transcription of step (c) and before the nick sealing of step (d).
The method according to any one of the preceding claims, wherein the DNA probe(s) provided in step (b) are non-phosphorylated, and the combination of steps (c) and (d) where the reverse transcription and nick translation is conditional for probe activation, before nick sealing can occur.
The method according to any one of the preceding claims, wherein a plurality of stretches of unknown identity are profiled in the same experiment, wherein each stretch of unknown identity is flanked by stretches of known identity, and whereby, for each stretch of unknown identity, at least one DNA probe having a first and a second end is/are provided, which are designed to hybridize to the sequences of known identity flanking the nucleotide stretch of unknown identity, thereby allowing profiling of multiple mutations, insertions, deletions and/or single nucleotide variations.
The method according to any one of the preceding claims, wherein if the method is performed in situ, sequencing is performed in situ, such as by Sequencing by Synthesis (SBS), Sequencing by Ligation (SBL), Sequencing by Hybridisation (SBH), SOLiD sequencing or single base extension sequencing.
The method according to any one of the preceding claims, wherein the method is performed in vitro, sequencing is performed via NGS sequencing post extraction of DNA probe(s) from the RNA sample.
The method according to any one of the preceding claims, wherein if the method is performed in situ, the probes, either pre or post amplified, are extracted from the RNA sample and processed for NGS sequencing.
The method according to any one of the preceding claims, wherein the method is used for both in situ and in vitro applications of:
Dependencies are inferred from the claim text.