Single cell analysis by polymerase cycling assembly

摘要

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.

主权项

1. A method of determining by single cell analysis the nucleotide sequences of fusion products comprising a plurality of linked nucleic acid molecules encoding at least two chains of a T-cell or B-cell immune receptor of a patient, the method comprising the steps of:
(a) separating single T-cells or B-cells from a sample comprising T-cells or B-cells of the patient among a plurality of reactors each containing 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 first nucleic acid molecule encoding one chain of an immune receptor and one or more pairs of such outer and linking primers being specific for a second nucleic acid molecule encoding another chain of the immune receptor,wherein both of said nucleic acid molecules have recombined nucleic acid sequences of a B-cell encoding a B-cell receptor or portion thereof or both of said nucleic acid molecules have recombined nucleic acid sequences of a T-cell encoding a T-cell receptor or portion thereof, respectively; (b) performing a PCA reaction in the reactors to form a plurality of different kinds of fusion products comprising a plurality of different nucleic acid molecules encoding the immune receptor chains of the single T-cell or B-cell; (c) spatially isolating individual molecules of said fusion products; and (d) sequencing the spatially isolated fusion product molecules to obtain nucleotide sequences of fusion products comprising a plurality of linked nucleic acid molecules encoding at least two chains of a T-cell or B-cell immune receptor of a patient.