US 12,442,037 B2
Multiplex method for detecting different analytes in a sample
Andreas Geipel, Mettmann (DE); Christian Korfhage, Langenfeld (DE); and Frank Reinecke, Essen (DE)
Assigned to Resolve Biosciences GMBH, Monheim am Rhein (DE)
Filed by Resolve Biosciences GMBH, Monheim Am Rhein (DE)
Filed on Dec. 17, 2021, as Appl. No. 17/554,093.
Application 17/554,093 is a continuation of application No. PCT/EP2021/066620, filed on Jun. 18, 2021.
Application 17/554,093 is a continuation of application No. PCT/EP2021/066668, filed on Jun. 18, 2021.
Claims priority of provisional application 63/129,942, filed on Dec. 23, 2020.
Claims priority of provisional application 63/127,910, filed on Dec. 18, 2020.
Prior Publication US 2022/0205028 A1, Jun. 30, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6841 (2018.01); C12Q 1/682 (2018.01); C12Q 1/6876 (2018.01); G01N 1/30 (2006.01); G01N 33/569 (2006.01)
CPC C12Q 1/6841 (2013.01) [C12Q 1/682 (2013.01); C12Q 1/6876 (2013.01); G01N 1/30 (2013.01); G01N 33/56961 (2013.01); C12Q 2600/16 (2013.01)] 16 Claims
OG exemplary drawing
 
1. A kit for multiplex analyte encoding, comprising
(A) at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes binding to a different analyte, wherein the analyte is a nucleic acid and each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically bind to different sub-structures of the same analyte by hybridization to form double stranded segments, each analyte-specific probe comprising
(aa) a binding element(S) that specifically binds to one of the different analytes to be encoded by hybridization to form a double stranded segment, and
(bb) an identifier element (T) comprising a specific identifier sequence which identifies the analyte to be encoded, wherein the analyte-specific probes of a particular set of analyte-specific probes share a common identifier element (T), and
wherein the analyte-specific probes of a particular set of analyte-specific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T),
wherein the analyte-specific probes in each set of analyte-specific probes bind to the same analyte and comprise the same nucleotide sequence of the identifier element (T) which is specific to said analyte; and
(B) at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises:
(aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the specific identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set such that an identifier connector element (t) and an identifier element (T) form, under annealing conditions, a T/t double stranded segment having a T/t melting temperature, and
(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide wherein the translator element (c) is not specific to an analyte;
wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the identifier connect element (t); and
(C) one set of signal oligonucleotides per signal element, wherein the number of different sets of signal elements is lower than the number of different types of analytes, each signal oligonucleotide comprising:
(aa) a translator connector element (C) comprising a nucleotide sequence which is not specific to an analyte, and which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide such that a translator element (c) and a translator connector element (C) form, under annealing conditions, a C/c double stranded segment having a C/c melting temperature, and
(bb) a signal element, wherein the signal element for each signal oligonucleotide is a fluorescent label;
wherein the T/t melting temperature and the C/c melting temperature are lower than a temperature necessary to dissociate the S binding element from its analyte such that a T/t double stranded segment and a C/c double stranded segment melt under conditions where the S binding element remains bound to its analyte; wherein the kit further comprises;
(D) at least a set of non-signal connector oligonucleotides for binding to a particular identifier element (T) wherein each non-signal connector oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a specific identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.