Kuukasjärvi T, Tanner M, Pennanen S, Karhu R, Visakorpi T, Isola J
Department of Pathology, Tampere University Hospital, Finland.
Genes Chromosomes Cancer. 1997 Feb;18(2):94-101.
The standard comparative genomic hybridization (CGH) protocol relies on availability of macroscopic tumor samples, which do not contain too much interfering normal cells. Recently, CGH after universal amplification of genomic DNA with degenerate oligonucleotide primed PCR (DOP-PCR) has been used to detect genetic aberrations in microdissected tumor specimens. However, owing to the technical difficulties, CGH results of only few microdissected samples have so far been published. We have developed an improved protocol for DOP-PCR, which includes direct incorporation of fluorochrome-conjugated nucleotides into the PCR product. Among the four polymerase enzymes tested. ThermoSequenase gave the best yield, with PCR products ranging from 100-4,000 bp. A two-step PCR-procedure was used, consisting of a preamplification with low stringency conditions followed by amplification in more stringent conditions. The method was first validated by hybridizing DOP-PCR-amplified normal DNA against nick-translated reference DNA, which showed uniform amd even hybridization result for all chromosomes. Comparison of DOP-PCR CGH to conventional CGH in MCF-7 breast cancer cell line further indicated that genetic aberrations can be reliable detected after DOP-PCR amplification. The sensitivity of the DOP-PCR-CGH was tested by serial dilution of MCF-7 DNA. Fifty picograms of sample DNA (corresponding roughly to two MCF-7 cells) was sufficient for high quality CGH. Experiments with cells microdissected from intraductal breast cancer demonstrated that carcinoma cells from 1 to 2 ducts were sufficient for a successful DOP-PCR CGH analysis. We conclude that the improved DOP-PCR-CGH protocol provides a powerful tool to study genetic aberrations in different histological subpopulations of malignant as well as precancerous lesions. DOP-PCR also improves the success rate of conventional paraffin-block CGH, because a poor quality or a too low yield of extracted DNA can be compensated by universal DNA amplification by DOP-PCR.
标准的比较基因组杂交（CGH）方案依赖于宏观肿瘤样本的可用性，这些样本不能含有过多干扰性的正常细胞。最近，用简并寡核苷酸引物PCR（DOP-PCR）对基因组DNA进行通用扩增后的CGH已被用于检测显微切割肿瘤标本中的基因畸变。然而，由于技术困难，迄今为止仅发表了少数显微切割样本的CGH结果。我们开发了一种改进的DOP-PCR方案，该方案包括将荧光染料偶联的核苷酸直接掺入PCR产物中。在所测试的四种聚合酶中，ThermoSequenase的产量最高，PCR产物范围为100 - 4000 bp。采用两步PCR程序，包括在低严谨条件下进行预扩增，然后在更严谨的条件下进行扩增。该方法首先通过将DOP-PCR扩增的正常DNA与缺口平移的参考DNA杂交进行验证，结果显示所有染色体的杂交结果均一且均匀。在MCF-7乳腺癌细胞系中，将DOP-PCR CGH与传统CGH进行比较，进一步表明DOP-PCR扩增后可以可靠地检测到基因畸变。通过对MCF-7 DNA进行系列稀释来测试DOP-PCR-CGH的灵敏度。50皮克的样本DNA（大致相当于两个MCF-7细胞）足以进行高质量的CGH分析。对乳腺导管内癌显微切割细胞的实验表明，1至2个导管的癌细胞足以成功进行DOP-PCR CGH分析。我们得出结论，改进的DOP-PCR-CGH方案为研究恶性以及癌前病变的不同组织学亚群中的基因畸变提供了一个强大的工具。DOP-PCR还提高了传统石蜡块CGH的成功率，因为提取DNA质量差或产量过低可以通过DOP-PCR进行通用DNA扩增来弥补。
