| CPC C12Q 1/6841 (2013.01) [C12Q 1/6804 (2013.01); G01N 33/5308 (2013.01); G01N 33/53 (2013.01)] | 28 Claims |
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1. A method of detecting a protein and a nucleic acid molecule in a cell in situ, said method comprising:
i) contacting the cell with a first polynucleotide probe and binding the first polynucleotide probe to the nucleic acid molecule, wherein the first polynucleotide probe comprises a first primer binding sequence and a first barcode sequence, wherein the first barcode sequence is from a known set of barcodes associated with the nucleic acid molecule;
ii) contacting the cell with a specific binding reagent and binding the specific binding reagent to the protein, wherein the specific binding reagent comprises an oligonucleotide comprising a second primer binding sequence and a second barcode sequence associated with the specific binding reagent, wherein the second barcode sequence is from a known set of barcodes associated with the protein; and
iii) hybridizing a first sequencing primer to the first primer binding sequence, and hybridizing a second sequencing primer to the second primer binding sequence, wherein the first sequencing primer and the second sequencing primer are different sequences, and
iv) incorporating one or more modified nucleotides into the first sequencing primer with a polymerase to create a first extension strand, and detecting the one or more incorporated nucleotides in a first optically resolvable feature, and, after generating the first extension strand, incorporating one or more modified nucleotides into the second sequencing primer with a polymerase to create a second extension strand, and detecting the one or more incorporated nucleotides in a second optically resolvable feature.
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