公开(公告)号：US20230383343A1

公开(公告)日：2023-11-30

申请号：US18201854

申请日：2023-05-25

申请人： Miltenyi Biotec B.V. & Co. KG

发明人： Robert Pinard ,  Andreas Bosio ,  Thomas Rothmann ,  Seiyu Hosono

IPC分类号： C12Q1/6874 ,  C12Q1/686 ,  C12Q1/6841

CPC分类号： C12Q1/6874 ,  C12Q1/6841 ,  C12Q1/686

摘要： Microscopy imaging that allows for multiple mRNAs, proteins and metabolites to be spatially resolved at a subcellular level provides valuable molecular information which is a crucial factor for understanding tissue heterogeneity as for example within the tumor micro environment. The current invention describes a method (High Density-SUMI-Seq) which combines the use of Spatial Unique Molecular Identifier in situ localization and identification (by in situ sequencing or sequential fluorescence hybridization) of rolonies derived from rolling circle amplification of circular oligonucleotides and in vitro sequencing of target amplified RNA or DNA in combination with SUMI identification at a subcellular level with no optical diffraction limitation in the amount of amplified target information that can be analyzed per cell. Apart from amplified RNA or DNA, the High Density-SUMI-Seq method can also be applied using linear oligonucleotides to spatially resolve proteins and metabolites to provide multiomics results.

公开(公告)号：US20230407383A1

公开(公告)日：2023-12-21

申请号：US18210119

申请日：2023-06-15

申请人： Miltenyi Biotec B.V. & Co. KG

发明人： Robert Pinard ,  Seiyu Hosono

IPC分类号： C12Q1/6869 ,  C12Q1/6862

CPC分类号： C12Q1/6869 ,  C12Q1/6862

摘要： The invention is directed to a method to simultaneously obtain both the spatial location and sequence information of a target sequence with a higher resolution than the known technologies. The method comprises steps to spatially localize the mRNA expressed on a tissue by the use of a hybrid circular/linear DNA probe with an UMI and—after several amplification steps, the obtaining sequence information by NGS.

公开(公告)号：US20240110232A1

公开(公告)日：2024-04-04

申请号：US18373441

申请日：2023-09-27

申请人： Miltenyi Biotec B.V. & Co. KG

发明人： Robert Pinard ,  Seiyu Hosono ,  Reto Mueller ,  Emily Neil

IPC分类号： C12Q1/6841 ,  C12Q1/6874

CPC分类号： C12Q1/6841 ,  C12Q1/6874 ,  C12Q1/682

摘要： The invention is directed to a method to obtain the spatial location and sequence information of a target sequence of at least one m-RNA strand on a tissue sample comprising the steps



a. providing a linear probe, containing a) a binding region capable of binding to the at least one m-RNA strand and b) an anchor sequence comprising a UMI region located between a first and a second locator regions and c) a primer region;
b. hybridizing the linear probe with its binding region to the m-RNA strand;
c. complementing the linear probe using the m-RNA strand as template thereby obtaining a reversed transcribed c-DNA strand
d. hybridizing a locator molecule with its 3′ and 5′ ends to the first and second locator regions thereby creating a gap corresponding to the length of the UMI of the linear probe
e. Filling the gap in the locator molecule with nucleotides complementary to the UMI using a non-strand displacement enzyme thereby creating a circular template comprising a copy of the UMI region from the linear probe.
f. multiplying the circular template molecule by RCA on the tissue sample, starting from a primer region thereby creating a rolony
g. Sequencing at least the UMI portion of the rolonies thereby obtaining the spatial location of the m-RNA on the tissue
h. removing the reversed transcribed c-DNA strand from the tissue and dehybridizing the m-RNA strand thereby obtaining a single stranded c-DNA oligomer
i. providing the single stranded cDNA oligomer with a first and a second adaptor primer at the 3′ and 5′ ends obtaining a primed single stranded oligomer; amplification of the primed single stranded oligomer by PCR
j. Sequencing the amplified primed single stranded oligomer and linking the spatial information of the rolonies with the sequence information of the amplified primed single stranded oligomer via the UMI sequence

公开(公告)号：US20230313274A1

公开(公告)日：2023-10-05

申请号：US18190534

申请日：2023-03-27

申请人： Miltenyi Biotec B.V. & Co. KG

发明人： Michael Perbost ,  Robert Pinard ,  Seiyu Hosono ,  Emily Neil

IPC分类号： C12Q1/6813

CPC分类号： C12Q1/6813

摘要： The present invention is to provide a method for hybridization of a photo-responsive oligonucleotide to a nucleic acid by providing the nucleic acid with a complementary oligonucleotide, wherein the oligonucleotide functions as a starting point for a polymerase for nucleic acid synthesis characterized in that the photo-responsive oligonucleotide comprises at least two photo-responsive elements which change from a first to a second conformation upon irradiation with light thereby disabling or enabling the oligonucleotide hybridization. In addition to that, the current invention provides a method for spatially controlled oligonucleotide hybridization to specific sites by spatial illumination of areas of no interest, thus changing the oligonucleotide conformation to a non-binding state. The reversable hybridization of the oligonucleotide can be used for controlling several reactions such as rolling circle amplification and a sequencing reaction.

公开(公告)号：US20210403992A1

公开(公告)日：2021-12-30

申请号：US17345011

申请日：2021-06-11

申请人： Miltenyi Biotec B.V. & Co. KG

发明人： Robert Pinard ,  Seiyu Hosono

IPC分类号： C12Q1/6869 ,  C12Q1/6825

摘要： Microscopy imaging that allow for multiple mRNAs to be resolved at a single cell level provides valuable information regarding transcript amount and localization, which is a crucial factor for understanding tissue heterogeneity, the molecular development and treatment of diseases. The current invention describes a method (Fly FISH) which combined the use of padlock oligonucleotides as fluorescence in situ hybridization (FISH) probes for detection and sequencing targeted portion of RNA or cDNA transcript at a cellular level with less limitation in the amount of transcripts and the length of the sequence that can be analyzed. Padlocks probes containing various barcodes in their core are utilized both as FISH probes and also to capture RNA portion that can be sequenced. The same barcodes can be used to selectively prime a rolling circle amplification and amplify a subset of transcripts coming from a specific region that have been tagged as of interest during the probing steps.

公开(公告)号：US20230348961A1

公开(公告)日：2023-11-02

申请号：US18307406

申请日：2023-04-26

申请人： Miltenyi Biotec B.V. & Co. KG

发明人： Seiyu Hosono ,  Jimmy Adediran ,  Robert Pinard

IPC分类号： C12Q1/6853 ,  C12Q1/6874

CPC分类号： C12Q1/6853 ,  C12Q1/6874

摘要： The invention is directed to a method for obtaining the sequence information of a target sequence from a tissue comprising at least one RNA or c-DNA strand comprising two-fold RCA.

公开(公告)号：US20230323438A1

公开(公告)日：2023-10-12

申请号：US18131128

申请日：2023-04-05

申请人： Miltenyi Biotec B.V. & Co. KG

发明人： Thomas Rothmann ,  Robert Pinard ,  Seiyu Hosono ,  Andreas Bosio

IPC分类号： C12Q1/6841

CPC分类号： C12Q1/6841

摘要： Microscopy imaging that allow for multiple mRNAs, proteins and metabolites to be spatially resolved at a subcellular level provides valuable molecular information which is a crucial factor for understanding tissue heterogeneity as for example within the tumor micro environment. The current invention describes a method (High Density—SUMI-Seq) which combines the use of Spatial Unique Molecular Identifier in situ localization and identification (by in situ sequencing or sequential fluorescence hybridization) of rolonies derived from rolling circle amplification of circular oligonucleotides and in vitro sequencing of target captured RNA or DNA in combination with SUMI identification at a subcellular level with no optical diffraction limitation in the amount of captured target information that can be analyzed per cell. Apart from captured RNA or DNA, the High Density—SUMI-Seq method can also be applied using linear oligonucleotides to spatially resolve proteins and metabolites to provide multiomics results.