Single cell analysis by polymerase cycling assembly
View Patent ↗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.
1. 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.
2. The method. of claim 1 wherein said multiple reactors are aqueous micelles of a water-in-oil emulsion.
3. The method of claim 2 wherein said water-in-oil emulsion is generated by a microfluidics device.
4. The method of claim 1 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 plurality of target nucleic acids.
5. The method of claim 1 further comprising a step of lysing said single lymphocytes in said reactors prior to said step of performing a PCA reaction.
6. The method of claim 1 wherein said clonotype comprises a portion of an immunoglobulin heavy chain (IgH) or a TCR β chain.