Method of spatial sequencing of genes from tissue using padlocks with gaps on substrate

Patent metadata

Field	Value
Publication number	US-2022403462-A1
Application number	US-202217745977-A
Country	US
Kind code	A1
Filing date	May 17, 2022
Priority date	Jun 18, 2021
Publication date	Dec 22, 2022
Grant date	—

Abstract

The invention is directed to a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the stepsa. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial markerb. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomerc. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial markerd. removing sample form surfacee. hybridizing at least one oligonucleotide comprising 50-1000 nucleic acids with its 5′ and 3′ ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular templatef. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming roloniesg. obtaining the sequence information of the roloniesh. linking the spatial information of the sample with the sequence information of the rolonies.

First claim

What is claimed is:

1 . A method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand, comprising the steps
a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker; b. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomer; c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker; d. removing sample form surface; e. hybridizing at least one oligonucleotide comprising 50-1000 nucleic acids with its 5′ and 3′ ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular template; f. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming rolonies; g. obtaining the sequence information of the rolonies; and h. linking the spatial information of the sample with the sequence information of the rolonies.

2 . The method according to claim 1 , characterized in that the spacer units are selected from the group consisting of antibodies, Fab fragments of antibodies, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies or aptamers .

3 . The method according to claim 1 , characterized in that the spacer units are selected from the group consisting of oligonucleotides comprising at least 5 Thymine (poly-T) single molecules and wherein the single stranded oligomer bond to the at least one spacer unit is reverse transcribed into a c-DNA strand and wherein the mRNA strand is removed by denaturation.

4 . The method according to claim 1 , characterized in that hybridizing the at least one oligonucleotide to complementary parts of the at least one single stranded oligomer thereby creating a padlock unit with a gap between the 5′ and the 3′ end of the oligonucleotide and filling the gap of the padlock unit with complementary nucleic acids as target sequence and ligate them to generate the single strand circular template.

5 . The method according to claim 1 , characterized in that hybridizing the at least one oligonucleotide to complementary parts of the at least one single stranded oligomer and ligate the 5′ and the 3′ end of the oligonucleotide to generate the single strand circular template, wherein the complementary parts of the at least one single stranded oligomer define the target sequence.

6 . The method according to claim 1 , characterized in that the spacer units are randomly distributed on the substrate.

7 . The method according to claim 1 , characterized in that the sample is permeabilized after providing to the surface.

8 . The method according to claim 1 , characterized in that in step d) the sample is removed form surface enzymatically or chemically.

9 . The method according to claim 1 , characterized in that the oligonucleotide comprises at least one primer sequence for the polymerase capable of rolling circle amplification.

10 . The method according to claim 1 , characterized in that the oligonucleotide is provided with at least one primer sequence for the polymerase capable of rolling circle amplification by ligation of a primer oligonucleotide.

11 . The method according to claim 1 , characterized in that the spatial location of the sample and the spatial location of the sequenced rolonies are superimposed relative to the location of the fiducial marker.

12 . The method according to claim 1 , characterized in that after providing the sample to the surface, the sample is stained to obtain the spatial location relative to the fiducial markers.

13 . The method according to claim 1 , characterized in that the sequencing information is obtained by sequencing by synthesis process

Assignees

• Miltenyi Biotec Bv & Co Kg

Inventors

• Meyer Hansueli
• Pinard Robert

Classifications

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• G16B20/50

  Mutagenesis · CPC title