Single cell analysis by polymerase cycling assembly

A method of distinguishing by single cell analysis multiple subpopulations of a population of lymphocytes, the method comprising the steps of;

• providing multiple reactors each containing a single lymphocyte of the population 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 outer and linking primers being specific for a nucleic acid containing a clonotype and one or more pairs of such outer and linking primers being specific for a plurality of target nucleic acids which is characteristic of a subpopulation of lymphocytes, wherein the clonotype is a recombined nucleic acid sequence of a B cell encoding a B cell receptor or portion thereof, and/or a T cell encoding a T cell receptor or portion thereof, respectively;
• performing a PCA reaction in each reactor to form a plurality of different kinds of fusion products comprising a plurality of different target nucleic acids and a clonotype of the single lymphocyte therein;
• sequencing the fusion products from the reactors;
• and
• classifying each single lymphocyte into a subpopulation by the target nucleic acids associated with its clonotype.