US 12,668,838 B2Granted

Gene specific spatial rolling circle amplification and NGS sequencing

AssigneeMiltenyi Biotec GmbH

InventorsRobert Pinard, Seiyu Hosono

Life of this patent

Estimating the term…

Abstract

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 a UMI and, after several amplification steps, obtaining the sequence information by NGS.

Claims8

Dependent claims

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 locator probe, the locator probe comprising:
i) a linear locator having a locator anchor region and an RNA anchor region capable of binding to the at least one m-RNA strand;
and
ii) a circular locator having a primer sequence with a first and a second primer region, a UMI region, and a region complementary to the locator anchor region of the linear locator;
b. hybridizing the RNA anchor region of the locator probe to the m-RNA strand;
c. extending the RNA anchor region of the locator probe using the m-RNA strand as template thereby obtaining a reverse transcribed c-DNA strand;
d. complementing the circular locator starting from the first primer region using the circular locator as template and ligating the resulting oligomer with the locator anchor region of the linear locator thereby obtaining an extended reverse transcribed c-DNA strand comprising the UMI region and the target sequence of the m-RNA strand;
e. multiplying the circular locator by RCA starting from the second primer region on the tissue sample creating at least one first rolony;
f. performing spatial resolution sequencing on the at least one first rolony thereby obtaining the spatial and sequence information of the at least one first rolony;
g. removing the extended reverse transcribed c-DNA strand from the tissue and de-hybridizing the extended reverse transcribed c-DNA strand from the m-RNA strand obtaining a single stranded oligomer;
h. providing the single stranded oligomer with a first and a second adaptor primer at the 3′ and 5′ ends obtaining a primed single stranded oligomer, amplifying the primed single stranded oligomer by PCR and circularizing the primed single stranded oligomer by ligation of the first and second adaptor primer with each other thereby creating a circular single stranded oligomer;
and
i. multiplying the circular single stranded oligomer by RCA into second rolonies;
sequencing the second rolonies and linking the spatial information of the first rolonies with the sequence information of the second rolonies via the UMI sequence.

11 more elements

Dependencies are inferred from the claim text.

patentgrep

Loading patent…

Abstract

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 locator probe, the locator probe comprising:
i) a linear locator having a locator anchor region and an RNA anchor region capable of binding to the at least one m-RNA strand;
and
ii) a circular locator having a primer sequence with a first and a second primer region, a UMI region, and a region complementary to the locator anchor region of the linear locator;
b. hybridizing the RNA anchor region of the locator probe to the m-RNA strand;
c. extending the RNA anchor region of the locator probe using the m-RNA strand as template thereby obtaining a reverse transcribed c-DNA strand;
d. complementing the circular locator starting from the first primer region using the circular locator as template and ligating the resulting oligomer with the locator anchor region of the linear locator thereby obtaining an extended reverse transcribed c-DNA strand comprising the UMI region and the target sequence of the m-RNA strand;
e. multiplying the circular locator by RCA starting from the second primer region on the tissue sample creating at least one first rolony;
f. performing spatial resolution sequencing on the at least one first rolony thereby obtaining the spatial and sequence information of the at least one first rolony;
g. removing the extended reverse transcribed c-DNA strand from the tissue and de-hybridizing the extended reverse transcribed c-DNA strand from the m-RNA strand obtaining a single stranded oligomer;
h. providing the single stranded oligomer with a first and a second adaptor primer at the 3′ and 5′ ends obtaining a primed single stranded oligomer, amplifying the primed single stranded oligomer by PCR and circularizing the primed single stranded oligomer by ligation of the first and second adaptor primer with each other thereby creating a circular single stranded oligomer;
and
i. multiplying the circular single stranded oligomer by RCA into second rolonies;
sequencing the second rolonies and linking the spatial information of the first rolonies with the sequence information of the second rolonies via the UMI sequence.

11 more elements