Methods of identifying multiple epitopes in cells

Abstract

The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.

1 . A method for identifying whether at least one mRNA target is present in a plurality of cells comprising:
 a) binding to the target mRNA in the plurality of cells at least one pair of SNAIL oligonucleotide primers comprising a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of the SPO and the PO comprise first complementarity regions (CR1 and CR1′ respectively), complementary to adjacent sequences on the target mRNA; and each of the SPO and the PO further comprise second complementarity regions (CR2 and CR2′, respectively) complementary to each other, wherein CR2′ hybridizes to CR2;   b) contacting the plurality of cells with ligase wherein the 5′ and the 3′ ends of the PO are joined to generate a closed circle;   c) performing rolling circle amplification using the PO as a template and SPO as a primer for a polymerase to form multiple copies of the PO template;   d) annealing an anchor primer complementary to a region of the PO to the multiple copies of the PO and forming an extension product which is comprises a copy of the CR1′;   e) forming a cell originating barcode (COB) by adding multiple assayable polymer subunit (APS) oligonucleotides to each extension product in the plurality of cells in an ordered manner during successive rounds of split pool synthesis wherein the APS oligonucleotides in each round anneal adjacently to the APS from a previous round via an annealing region, and covalently linking the adjacently annealed APS oligonucleotides to each other to create unique codes that represent the identities of individual cells in which the SNAIL primers are bound to targets.   
     
     
         2 . The method of  claim 1 , wherein the APSs anneal to a splint oligonucleotide annealed to the anchor prior to being covalently linked to each other. 
     
     
         3 . The method of  claim 1 , wherein the APS oligonucleotides are linked to each other by Click chemistry. 
     
     
         4 . The method of  claim 1 , wherein the APS oligonucleotides are linked to each other by ligation. 
     
     
         5 . The method of  claim 4 , wherein the ligation is preceded by gap filling. 
     
     
         6 . The method of  claim 1 , wherein a plurality of SNAIL oligonucleotide primer pairs having specificity for different target mRNAs [ claim 1 ] are used. 
     
     
         7 . The method of  claim 2 , wherein the splint comprises a sample identification barcode. 
     
     
         8 . The method of  claim 1 , further comprising a step for sequencing the COB and the copy of the CR1′ thus identifying the individual cells wherein the SNAIL primer pairs are bound to the target sequence. 
     
     
         9 . The method of  claim 8 , wherein the COB and the copy of the CR1′ are amplified prior to sequencing. 
     
     
         10 . The method of  claim 9 , wherein the splint comprises a pair of primer binding regions for the amplification. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the anchor comprises an Epitope Specific Barcode (ESB) sequence. 
     
     
         13 . The method of  claim 1 , wherein CR2′ is a split region such that in step a), the 5′ and the 3′ ends of the PO hybridize to CR2 adjacent to one another. 
     
     
         14 . The method of  claim 1 , wherein CR2′ is a split region such that in step a), the 5′ and the 3′ ends of the PO hybridize to CR2 leaving a gap and prior to ligation in step b), the plurality of cells are contacted with a DNA polymerase. 
     
     
         15 . The method of  claim 1 , wherein CR1 and CR1′ are complementary to adjacent sequences on the target mRNA separated by no more than 10 nucleotides. 
     
     
         16 . A kit for identifying whether a plurality of mRNA targets are present in a plurality of cells, the kit comprising:
 a) one or more pairs of SNAIL oligonucleotide primers comprising a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of SPO and PO comprise first complementarity regions (CR1 and CR1′ respectively), complementary to adjacent sequences on the target mRNA; and each of SPO and PO further comprise second complementarity regions (CR2 and CR2′, respectively) complementary to each other;   b) an anchor primer complementary to a region of the PO;   c) a splint oligonucleotide capable of annealing to the anchor; and   d) a plurality of assayable polymer subunit (APS) oligonucleotides capable of annealing to the split in a ordered manner during successive rounds of annealing via round-specific hybridization regions.   
     
     
         17 . The kit of  claim 15 , further comprising one or more of the following: DNA polymerase, DNA ligase, dNTPs, and primer pairs complementary to the primer binding regions in the splint. 
     
     
         18 . The kit of  claim 16 , wherein the DNA polymerase includes one or more of strand displacing DNA polymerase and a DNA polymerase suitable for PCR. 
     
     
         19 . The kit of  claim 17 , wherein the DNA polymerase includes PhiX29 DNA polymerase, T4 DNA polymerase and a DNA polymerase suitable for PCR. 
     
     
         20 . The kit of  claim 15 , further comprising reagents for immobilization of cell originating barcodes (COB).