Single cell analysis by polymerase cycling assembly

Abstract

The invention provides a method of making measurements on individual cells of a population, particularly cells that have identifying nucleic acid sequences, such as lymphoid cells. In one aspect, the invention provides a method of making multiparameter measurements on individual cells of such a population by carrying out a polymerase cycling assembly (PCA) reaction to link their identifying nucleic acid sequences to other cellular nucleic acids of interest. The fusion products of such PCA reaction are then sequenced and tabulated to generate multiparameter data for cells of the population.

What is claimed is: 
     
         1 . A method of analyzing a plurality target nucleic acids in each cell of a population, the method comprising the steps of:
 providing multiple reactors each containing a single cell in a polymerase cycling assembly (PCA) reaction mixture comprising a pair of outer primers and one or more pairs of linking primers specific for the plurality of target nucleic acids;   performing a PCA reaction in the reactors to form fusion products of the target nucleic acids in the reactors; and   sequencing the fusion products from the reactors to identify the target nucleic acids of each cell in the population.   
     
     
         2 . The method of  claim 1  wherein said multiple reactors are aqueous micelles of a water-in-oil emulsion. 
     
     
         3 . The method of  claim 3  wherein said water-in-oil emulsion is generated by a microfluidics device. 
     
     
         4 . The method of  claim 1  wherein said population is a population of B cells and/or T cells. 
     
     
         5 . The method of  claim 4  wherein at least one pair of primers from said outer primers and said linking primers is specific for a clonotype of said B cells and/or T cells. 
     
     
         6 . The method of  claim 5  wherein at least one pair of primers from said outer primers and said linking primers is specific for a nucleic acid sequence of said B cells and/or T cells that is a cancer marker or encodes a cancer marker. 
     
     
         7 . The method of  claim 6  wherein said nucleic acid is an RNA that indicates a cancerous state by over expression. 
     
     
         8 . The method of  claim 6  wherein said nucleic acid is a DNA that indicates a cancerous state by excess copy number. 
     
     
         9 . A method of distinguishing multiple subpopulations of lymphocytes, the method comprising the steps of:
 providing multiple reactors each containing a single lymphocyte in a polymerase cycling assembly (PCA) reaction mixture comprising a pair of outer primers and one or more pairs of linking primers, at least one pair of such primers being specific for a nucleic acid containing a clonotype and one or more pairs of such primers being specific for one or more target nucleic acids characteristic of the multiple subpopulations of lymphocytes;   performing a PCA reaction in each reactor to form a fusion product comprising the target nucleic acids and a clonotype of the lymphocyte therein;   sequencing the fusion products from the reactors; and   classifying each lymphocyte into a subpopulation by the target nucleic acids associated with its clonotype.   
     
     
         10 . The method of  claim 9  wherein said multiple reactors are aqueous micelles of a water-in-oil emulsion. 
     
     
         11 . The method of  claim 10  wherein said water-in-oil emulsion is generated by a microfluidics device. 
     
     
         12 . The method of  claim 9  wherein said nucleic acid containing said clonotype and said one or more target nucleic acids are RNA and wherein said step of classifying includes determining the relative expression levels of said one or more target nucleic acids. 
     
     
         13 . A method of detecting cross-lineage rearrangements in a population of lymphocytes, the method comprising the steps of:
 providing multiple reactors each containing a single lymphocyte in a polymerase cycling assembly (PCA) reaction mixture comprising a pair of outer primers and one or more pairs of linking primers, at least one pair of such primers being specific for a nucleic acid containing at least a portion of a B cell receptor gene and at least one pair of such primers being specific for a nucleic acid containing at least a portion of a T cell receptor gene;   performing a PCA reaction in each reactor to form a fusion product comprising the target nucleic acids and a clonotype of the lymphocyte therein;   sequencing the fusion products from the reactors; and   determining the presence, absence or level of fusion products that comprise both a portion of a B cell receptor gene and a portion of a T cell receptor gene to detect cross-lineage rearrangements in the population of lymphocytes.   
     
     
         14 . The method of  claim 13  wherein said multiple reactors are aqueous micelles of a water-in-oil emulsion. 
     
     
         15 . The method of  claim 14  wherein said water-in-oil emulsion is generated by a microfluidics device.