Amplification and detection of a Y-chromosome DNA sequence by fluorescence in situ polymerase chain reaction and flow cytometry using cells in suspension.
Amplification and detection of a Y-chromosome DNA sequence by fluorescence in situ polymerase chain reaction and flow cytometry using cells in suspension.
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DOI：
10.1002/cyto.990220313
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发表时间：
1995-09
期刊：
影响因子：
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通讯作者：
E. A. Timm;E. Podniesinski;L. Duckett;J. Cardott;C. Stewart
中科院分区：
文献类型：
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作者：
E. A. Timm;E. Podniesinski;L. Duckett;J. Cardott;C. Stewart
利用悬浮细胞和流式细胞术建立了一种原位扩增和检测核酸序列的方法。这一过程涉及使用聚合酶链式反应(PCR)和我们实验室开发的荧光原位杂交(FISH)方法来检测扩增的PCR产物。在这些研究中，我们扩增了Y染色体特异的重复DNA序列。以男性建立的B细胞淋巴瘤细胞系Daudi细胞为阳性对照，女性建立的早幼粒白血病细胞系HL-60为阴性对照。在原位聚合酶链式反应过程中，细胞自身荧光(噪声)增加，导致与阳性细胞内扩增产物结合的探针(信号)的检测灵敏度显着降低。应用了集成到AS标准台式流式细胞仪中的自体荧光还原电路来降低这种噪声，从而使信号的检测灵敏度提高了10倍。在没有应用自发荧光还原电路的情况下，阳性对照直方图分布与阴性对照样本分布几乎无法区分。自体荧光减少后，数据显示，只有在完全原位聚合酶链式反应的Daudi细胞中扩增出Y染色体DNA。这种提高的灵敏度也提供了对Y染色体重复序列的直接检测，尽管与原位聚合酶链式反应后扩增的目标相比，显示的信号较少。
A procedure for amplifying and detecting nucleic acid sequences in situ using cells in suspension and flow cytometry has been developed. The process involves the use of the polymerase chain reaction (PCR) and a fluorescent in situ hybridization (FISH) protocol developed in our laboratory to detect the amplified PCR product. For these studies, a Y-chromosome specific repeat DNA sequence was amplified. Daudi cells, a B-cell lymphoma culture line established from a male, was used as a positive control and HL-60, a promyelocytic leukemia culture line established from a female, was used as a negative control. During the in situ PCR process cellular autofluorescence (noise) increases causing markedly reduced detection sensitivity of the probe (signal) bound to the amplified product within the positive cells. An autofluorescence reduction circuit was applied which was integrated into as standard bench top flow cytometer to reduce this noise, thereby producing a 10-fold increase in detection sensitivity of the signal. Without the application of the autofluorescence reduction circuit, the positive control histogram distribution was virtually indistinguishable from the negative control sample distributions. After autofluorescence reduction, the data showed that the Y-chromosome DNA was only amplified in the Daudi cells subjected to the complete in situ PCR protocol. This increased sensitivity also provided direct detection of the Y-chromosome repeat sequence, albeit exhibiting less signal compared to the amplified target after the in situ PCR.