Modified double-stranded oligonucleotides that have terminal regions on each of their strands, that have a hybrid length of 6-50 nucleotides long, that have a melting temperature Tm of at least 32° C., and that include 2-4 modifying groups, each covalently attached to a different terminal region, preferably to a terminal nucleotide, said modifying groups being polycyclic substituents that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of binding to the 5′ exonuclease domains of DNA polymerases and, when included in a PCR or other primer-dependent DNA amplification reaction at a concentration, generally not more than 2000 nM, that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against 3′ terminal mismatches. increasing polymerase selectivity against AT-rich 3′ ends, reducing scatter among replicates, suppressing polymerase 5′ exonuclease activity, and inhibiting polymerase activity; as well as amplification reaction mixtures containing such modified double-stranded oligonucleotides, and amplification reactions, amplification assays and kits that include such modified double-stranded oligonucleotides.

Modified double-stranded oligonucleotides that have terminal regions on each of their strands, that have a hybrid length of 6-50 nucleotides long, that have a melting temperature Tm of at least 32° C., and that include 2-4 modifying groups, each covalently attached to a different terminal region, preferably to a terminal nucleotide, said modifying groups being polycyclic substituents that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of binding to the 5′ exonuclease domains of DNA polymerases and, when included in a PCR or other primer-dependent DNA amplification reaction at a concentration, generally not more than 2000 nM, that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against 3′ terminal mismatches. increasing polymerase selectivity against AT-rich 3′ ends, reducing scatter among replicates, suppressing polymerase 5′ exonuclease activity, and inhibiting polymerase activity; as well as amplification reaction mixtures containing such modified double-stranded oligonucleotides, and amplification reactions, amplification assays and kits that include such modified double-stranded oligonucleotides.

Provided herein are methods of detecting an analyte of interest to interrogate spatial gene expression in a sample.

Provided herein are methods of detecting an analyte of interest to interrogate spatial gene expression in a sample.

The invention is directed to a method to obtain the spatial location and sequence information of at least a part of a RNA or cDNA strand (006) in a sample comprising the steps a. hybridizing a first detection probe oligonucleotide (204) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the at least one RNA or cDNA strand, wherein the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide (205) comprising 5-100 nucleotides wherein a gap region (206) capable of binding oligonucleotides is created b. filling the gap region (206) in part with 1 to 16 barcode oligonucleotides comprising 4-20 nucleotides, wherein the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample c. partially hybridizing a second detection probe oligonucleotide (204′) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the same or cDNA strand and with the respective other end to the bridge oligonucleotide (205) to create a circular template d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers e. determining the sequence of nucleotides of the rolonies

The invention is directed to a method to obtain the spatial location and sequence information of at least a part of a RNA or cDNA strand (006) in a sample comprising the steps a. hybridizing a first detection probe oligonucleotide (204) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the at least one RNA or cDNA strand, wherein the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide (205) comprising 5-100 nucleotides wherein a gap region (206) capable of binding oligonucleotides is created b. filling the gap region (206) in part with 1 to 16 barcode oligonucleotides comprising 4-20 nucleotides, wherein the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample c. partially hybridizing a second detection probe oligonucleotide (204′) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the same or cDNA strand and with the respective other end to the bridge oligonucleotide (205) to create a circular template d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers e. determining the sequence of nucleotides of the rolonies

Provided herein are methods, compositions, and kits for preparing biological samples for multiplex spatial gene expression and proteomic analysis, such as determining a location of a nucleic acid analyte and a protein analyte in a biological sample.

Provided herein are methods, compositions, and kits for preparing biological samples for multiplex spatial gene expression and proteomic analysis, such as determining a location of a nucleic acid analyte and a protein analyte in a biological sample.

Provided is multiplex and asymmetric amplification of nucleic acid molecules. In particular, provided is a method for simultaneous and asymmetric amplification of one or more target nucleic acids in a sample. The method can simultaneously and asymmetrically amplify multiple target nucleic acids existing in a sample, and can simultaneously produce large number of single stranded products.

Provided is multiplex and asymmetric amplification of nucleic acid molecules. In particular, provided is a method for simultaneous and asymmetric amplification of one or more target nucleic acids in a sample. The method can simultaneously and asymmetrically amplify multiple target nucleic acids existing in a sample, and can simultaneously produce large number of single stranded products.