Improving degenerate oligonucleotide primed PCR‐comparative genomic hybridization for analysis of DNA copy number changes in tumors
Improving degenerate oligonucleotide primed PCR‐comparative genomic hybridization for analysis of DNA copy number changes in tumors
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改进简并寡核苷酸引发的 PCR 比较基因组杂交，用于分析肿瘤中 DNA 拷贝数的变化
DOI：
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发表时间：
2000
期刊：
影响因子：
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通讯作者：
R. Chaganti
中科院分区：
文献类型：
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作者：
Qiang Huang;S. Schantz;P. Rao;J. Mo;S. Mccormick;R. Chaganti
简并寡核苷酸启动的聚合酶链式反应(DOP-PCR)与比较基因组杂交(CGH)的结合使分析单细胞基因组变化成为可能。虽然DOP-PCR-CGH方法已有报道，但该方法的重复性一直不确定。我们通过系统地评价不同的标记方法(包括缺口平移、PCR掺入和随机标记)和不同的杂交混合物(包括扩增的检测DNA与扩增的参考DNA，称为同源杂交；以及扩增的检测DNA与未扩增的参考DNA，反之亦然，称为异源杂交)，建立了一种可重复性的DOP-PCR-CGH方法。我们用不同标记的探针和杂交组合分析了新鲜/冷冻的正常组织和肿瘤组织、福尔马林固定和石蜡包埋的肿瘤组织以及肿瘤细胞系等16个组织来源的DNA样本，并计算了DOP-PCR-CGH与标准CGH的符合率(CR)。我们发现，与标准CGH相比，同源杂交产生了高CRs的重复性结果(91-100%CR，平均97%)；相反，异源杂交未能产生低CRS(57-97%CR，平均80%；χ2=1245.8，P<；0.0001)、高背景、杂交不均匀和错误缺失或扩增的可重复性杂交结果。此外，我们改进的DOP-PCR方法将扩增效率提高了至少五倍，与以前报道的方法相比，允许在短短12.5pg的起始DNA中检测到基因组不平衡。综上所述，DOP-PCR-CHG同源杂交法，特别是与缺口平移标记相结合的方法，是可靠和重复性好的。该方法可用于利用微量的肿瘤DNA筛选基因组失衡，从而促进CGH的应用。基因染色体癌28：395-403,2000。©2000 Wiley-Liss Inc.
Combining degenerate oligonucleotide‐primed PCR (DOP‐PCR) with comparative genomic hybridization (CGH) has made it possible to analyze genomic changes in single cells. Although DOP‐PCR‐CGH methodology has been reported, the reproducibility of the method has been uncertain. We have developed a reproducible DOP‐PCR‐CGH protocol by systematically evaluating different labeling methods (including nick translation, PCR incorporation, and random‐primed labeling) and different hybridization mixtures (including amplified test DNA vs. amplified reference DNA, termed homo‐hybridization; and amplified test DNA vs. unamplified reference DNA or vice versa, termed hetero‐hybridization). We have analyzed DNA samples obtained from 16 tissue sources including fresh/frozen normal and tumor samples, formalin fixed and paraffin embedded tumor tissue, and tumor cell lines by using differently labeled probes and hybridization combinations, and we calculated the corresponding rate (CR) of DOP‐PCR‐CGH with standard CGH. We found that homo‐hybridization produced reproducible results with high CRs as compared to standard CGH (91–100% CR, mean 97%); In contrast, hetero‐hybridization failed to generate reproducible hybridization with low CRs (57–97% CR, mean 80%; χ2 = 1245.8, P < 0.0001), high background, uneven hybridization, and false deletions or amplifications. In addition, our improved DOP‐PCR protocol raised the amplification efficiency at least five times as compared to previously reported protocols, allowing for the detection of genomic imbalances in as little as 12.5 pg of starting DNA. In conclusion, the DOP‐PCR‐CHG homo‐hybridization method, especially when combined with labeling by nick translation, is reliable and reproducible. The method can be used in screening for genomic imbalances using minute amounts of tumor DNA, thereby facilitating CGH application. Genes Chromosomes Cancer 28:395–403, 2000. © 2000 Wiley‐Liss, Inc.
影响因子：
20.3
作者：
Rao, PH;Houldsworth, J;Chaganti, RSK
通讯作者：
Chaganti, RSK