AMPLIFICATION OF GENES, SINGLE TRANSCRIPTS AND CDNA LIBRARIES FROM ONE-CELL AND DIRECT-SEQUENCE ANALYSIS OF AMPLIFIED PRODUCTS DERIVED FROMONE MOLECULE

We report a procedure to generate and amplify cDNA libraries and to am plify and sequence genes and single RNA transcript molecules from the same cell without cloning. An absence of cloning steps minimizes poten tial sources of contamination, which can be especially problematic whe n working at the single cell level. Potential contamination is further reduced by an absence of any purification step prior to PCR amplifica tion. Amplifications are designed to minimize the production of aberra nt molecules in favor of full-length products, which is especially adv antageous when generating cDNA libraries. Genes are amplified from iso lated single nuclei, which are segregated from cytoplasmic lysates by microcentrifugation. Specific cDNA, total cDNA or both are synthesized from aliquots of the cytoplasmic lysate, and single cDNA molecules ar e isolated from others of the same species by limiting dilution prior to PCR amplification. In this way, the frequency of amplified products provides for a direct calculation of cDNA copy number by a Poisson an alysis, Incorporation errors by Tag DNA polymerase occur at a low freq uency and can be eliminated by sequencing independently amplified cDNA molecules from the same cell. Single molecule amplifications provide sufficient material for numerous (similar to 150) direct DNA sequencin g reactions. The limiting dilution approach also permits sequence info rmation to be obtained from a single cDNA, when highly related transcr ipts derived from distinct genes are present in the same cell and simu ltaneously amplified with the same primers. In sum, this method provid es for a maximum amount of nucleic acid information to be extracted fr om one cell. It has a wide range of applications to studies of the imm une system where, to a first approximation, each lymphocyte has a uniq ue receptor identity, where specific states of differentiation may be difficult to assess in a mixed cell population, and where cell immorta lization procedures are not always possible nor practical.