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本文报告了一种用多聚酶链反应(PCR)在骨髓或外周血常规涂片上,对单个细胞内的特定核酸片段进行原位扩增的方法。该法要点是:将细胞玻片切小,放入PCR反应管内,再加入100μl反应液,液内含特定引物,0.5U的Taq多聚酶和终浓度为1μci/100μl的放射性标记H3CTP,按95℃ 2分钟、54℃1分种、72℃ 2分种进行PCR。30周期后,以1×SSC、0.1×SSC先后各洗涤10分钟,然后用柯达核示踪胶乳覆盖玻片,暗处曝光24小时显影,继以姬姆萨染液复染,显微镜照相。
Here we report a method for the in situ amplification of specific nucleic acid fragments in a single cell by polymerase chain reaction (PCR) on conventional smears of bone marrow or peripheral blood. The method points are: small cell slides cut into PCR reaction tube, then add 100μl reaction solution containing specific primers, 0.5U of Taq polymerase and final concentration of 1μci / 100μl of radioactive labeled H3CTP, according to 95 ℃ 2 minutes, 54 ℃ 1 minute, 72 ℃ 2 minute PCR. After 30 cycles, each was washed for 10 minutes with 1 x SSC and 0.1 x SSC, and then covered with Kodak nuclear tracer latex, exposed for 24 hours in the dark, counterstained with a Giemsa stain, and photographed under a microscope.