Larramendy M L, El-Rifai W, Knuutila S
Department of Medical Genetics, Haartman Institute, University of Helsinki, Finland.
Cytometry. 1998 Mar 1;31(3):174-9.
In this study, we investigated whether fluorescein isothiocyanate (FITC)-labeling of test DNA and Texas-red (TR) labeling of reference DNA in comparative genomic hybridization (CGH) experiments cause the results to differ from those obtained using the opposite combination (reverse labeling). Analysis was performed on a total of 20 DNA specimens consisting of 13 frozen bone marrow aspirates from patients with acute myeloid leukemia, and fresh peripheral blood samples from seven healthy donors. For CGH, one aliquot from each test DNA sample was labeled using nick-translation with FITC-dUTP and another with TR-dUTP. Afterwards, the FITC-dUTP and TR-dUTP-labeled test DNAs were hybridized to TR-dUTP- and FITC-dUTP-labeled normal reference DNAs, respectively. The results using the two combinations were compared with each other and with the results of G-banding karyotype analysis. Karyotype data was used to detect artifacts known to occur in some chromosome regions in CGH analysis. The control DNAs labeled with FITC or TR showed no DNA copy number changes. Regardless of the fluorochrome employed for labeling, no DNA copy number changes were detected using CGH in patients with normal karyotypes, nor in patients whose karyotype aberrations were present in less than 40% of cells. In the remaining patients, CGH revealed DNA copy number changes that coincided with the results of the G-banding analysis. Hybridization artifacts known to occur in CGH experiments affecting chromosome regions 1p33-pter, 16p, 17p, 19, and 22 were observed in 15-23% of the tumor samples labeled with FITC, but not in samples labeled with TR. In addition, other previously unreported overrepresentations affecting 7q21, 9q34, 16q, 17q, and chromosome 20 were observed at very low frequencies in up to 10% of the samples when FITC was used to label test DNA. However, when TR was used, overrepresentations were observed at 4q13-q21, 11q21-q23, 13q21-qter, and Xq21-q22, whereas 19p was underrepresented. The results demonstrate that TR-labeling confirms abnormalities detected using FITC-labeling and reduces hybridization artifacts in the known problematic regions of the human genome.
在本研究中，我们调查了在比较基因组杂交（CGH）实验中，用异硫氰酸荧光素（FITC）标记测试DNA以及用德克萨斯红（TR）标记参照DNA是否会导致结果与使用相反组合（反向标记）所获得的结果不同。对总共20个DNA样本进行了分析，其中包括13例急性髓系白血病患者的冷冻骨髓穿刺物，以及7名健康供者的新鲜外周血样本。对于CGH，每个测试DNA样本的一份等分试样使用FITC-dUTP通过切口平移进行标记，另一份用TR-dUTP标记。之后，将FITC-dUTP和TR-dUTP标记的测试DNA分别与TR-dUTP和FITC-dUTP标记的正常参照DNA进行杂交。将两种组合的结果相互比较，并与G显带核型分析的结果进行比较。核型数据用于检测CGH分析中已知在某些染色体区域出现的假象。用FITC或TR标记的对照DNA未显示DNA拷贝数变化。无论用于标记的荧光染料如何，在核型正常的患者以及核型异常细胞比例低于40%的患者中，使用CGH均未检测到DNA拷贝数变化。在其余患者中，CGH揭示的DNA拷贝数变化与G显带分析结果一致。在15%至23%用FITC标记的肿瘤样本中观察到了CGH实验中已知会影响染色体区域1p33 - pter、16p、17p、19和22的杂交假象，但在用TR标记的样本中未观察到。此外，当用FITC标记测试DNA时，在高达10%的样本中以非常低的频率观察到了其他先前未报道的影响7q21、9q34、16q、17q和20号染色体的过度代表情况。然而，当使用TR时，在4q13 - q21、11q21 - q23、13q21 - qter和Xq21 - q22观察到过度代表情况，而19p则呈现低代表情况。结果表明，TR标记证实了使用FITC标记检测到的异常，并减少了人类基因组已知问题区域中的杂交假象。
