Jena P K, Liu A H, Smith D S, Wysocki L J
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206, USA.
J Immunol Methods. 1996 Apr 19;190(2):199-213. doi: 10.1016/0022-1759(95)00277-4.
We report a procedure to generate and amplify cDNA libraries and to amplify and sequence genes and single RNA transcript molecules from the same cell without cloning. An absence of cloning steps minimizes potential sources of contamination, which can be especially problematic when working at the single cell level. Potential contamination is further reduced by an absence of any purification step prior to PCR amplification. Amplifications are designed to minimize the production of aberrant molecules in favor of full-length products, which is especially advantageous when generating cDNA libraries. Genes are amplified from isolated 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 are 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 analysis. Incorporation errors by Taq DNA polymerase occur at a low frequency and can be eliminated by sequencing independently amplified cDNA molecules from the same cell. Single molecule amplifications provide sufficient material for numerous (approximately 150) direct DNA sequencing reactions. The limiting dilution approach also permits sequence information to be obtained from a single cDNA, when highly related transcripts derived from distinct genes are present in the same cell and simultaneously amplified with the same primers. In sum, this method provides for a maximum amount of nucleic acid information to be extracted from one cell. It has a wide range of applications to studies of the immune system where, to a first approximation, each lymphocyte has a unique receptor identity, where specific states of differentiation may be difficult to assess in a mixed cell population, and where cell immortalization procedures are not always possible nor practical.
我们报告了一种无需克隆即可从同一细胞中生成和扩增cDNA文库、扩增基因和单个RNA转录本分子并进行测序的方法。没有克隆步骤可将潜在的污染来源降至最低，这在单细胞水平操作时可能尤其成问题。在PCR扩增之前不存在任何纯化步骤，进一步减少了潜在的污染。扩增设计旨在尽量减少异常分子的产生，有利于全长产物的生成，这在生成cDNA文库时特别有利。基因从分离的单核中扩增，单核通过微量离心与细胞质裂解物分离。从细胞质裂解物的等分试样中合成特异性cDNA、总cDNA或两者，并且在PCR扩增之前通过有限稀释从同一物种的其他分子中分离单个cDNA分子。通过这种方式，扩增产物的频率可通过泊松分析直接计算cDNA拷贝数。Taq DNA聚合酶的掺入错误发生频率较低，可通过对来自同一细胞的独立扩增的cDNA分子进行测序来消除。单分子扩增为大量（约150个）直接DNA测序反应提供了足够的材料。当来自不同基因的高度相关转录本存在于同一细胞中并使用相同引物同时扩增时，有限稀释方法还允许从单个cDNA获得序列信息。总之，这种方法可从一个细胞中提取最大量的核酸信息。它在免疫系统研究中有广泛的应用，在免疫系统中，初步估计每个淋巴细胞都有独特的受体身份，在混合细胞群体中可能难以评估特定的分化状态，并且细胞永生化程序并不总是可行或实用的。
