The present invention relates to a digital multiplex method for detecting and/or quantifying multiple target biomolecules in a sample, said biomolecules being selected from DNA, RNA, and proteins. The present invention further relates to different applications of the digital multiplex method and to a kit.

The present invention relates to a digital multiplex method for detecting and/or quantifying multiple target biomolecules in a sample, said biomolecules being selected from DNA, RNA, and proteins. The present invention further relates to different applications of the digital multiplex method and to a kit.

Disclosed are methods for detecting a target nucleic acid in a sample. The methods include contacting the sample, in the presence of a polymerase and an endonuclease, with a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of a target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide.

Disclosed are methods for detecting a target nucleic acid in a sample. The methods include contacting the sample, in the presence of a polymerase and an endonuclease, with a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of a target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide.

Cleavable primers are incorporated into single cell analysis workflows to reduce and/or eliminate misprimed nucleic acid amplicons. Specifically, cleavable primers can introduce restriction endonuclease cleavage sites into misprimed nucleic acid amplicons. For example, cleavable primers can introduce a restriction endonuclease cleavage site into an amplicon comprising DNA misprimed by an RNA primer. As another example, cleavable primers can introduce a restriction endonuclease cleavage site into an amplicon comprising cDNA misprimed by a DNA primer. Such amplicons can then be cleaved by a restriction endonuclease to remove them from identification and association in subsequent nucleic acid sequencing.

Cleavable primers are incorporated into single cell analysis workflows to reduce and/or eliminate misprimed nucleic acid amplicons. Specifically, cleavable primers can introduce restriction endonuclease cleavage sites into misprimed nucleic acid amplicons. For example, cleavable primers can introduce a restriction endonuclease cleavage site into an amplicon comprising DNA misprimed by an RNA primer. As another example, cleavable primers can introduce a restriction endonuclease cleavage site into an amplicon comprising cDNA misprimed by a DNA primer. Such amplicons can then be cleaved by a restriction endonuclease to remove them from identification and association in subsequent nucleic acid sequencing.

A method for spatially tagging nucleic acids of a biological specimen, including steps of (a) providing a solid support comprising different nucleic acid probes that are randomly located on the solid support, wherein the different nucleic acid probes each includes a barcode sequence that differs from the barcode sequence of other randomly located probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequences on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located probes; (d) hybridizing the randomly located probes to target nucleic acids from portions of the biological specimen; and (e) modifying the randomly located probes that are hybridized to the target nucleic acids, thereby producing modified probes that include the barcode sequences and a target specific modification, thereby spatially tagging the nucleic acids of the biological specimen.

A method for spatially tagging nucleic acids of a biological specimen, including steps of (a) providing a solid support comprising different nucleic acid probes that are randomly located on the solid support, wherein the different nucleic acid probes each includes a barcode sequence that differs from the barcode sequence of other randomly located probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequences on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located probes; (d) hybridizing the randomly located probes to target nucleic acids from portions of the biological specimen; and (e) modifying the randomly located probes that are hybridized to the target nucleic acids, thereby producing modified probes that include the barcode sequences and a target specific modification, thereby spatially tagging the nucleic acids of the biological specimen.

The present disclosure provides compositions and methods for template-free double stranded geometric enzymatic nucleic acid synthesis of arbitrarily programmed nucleic acid sequences.

The present disclosure provides compositions and methods for template-free double stranded geometric enzymatic nucleic acid synthesis of arbitrarily programmed nucleic acid sequences.