Amplification of whole tumor genomes and geneby-gene mapping of genomic aberrations from limited sources of fresh-frozen and paraffin-embedded DNA
Amplification of whole tumor genomes and geneby-gene mapping of genomic aberrations from limited sources of fresh-frozen and paraffin-embedded DNA
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DOI：
10.1016/s1525-1578(10)60543-0
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发表时间：
2005-05-01
影响因子：
4.1
通讯作者：
Sikic, BI
中科院分区：
文献类型：
--
作者：
Bredel, M;Bredel, C;Sikic, BI
足够数量的基因组DNA可能成为临床组织样本全基因组分析的瓶颈。DNA聚合酶Phi29可以用于全基因组的随机启动扩增，尽管这种扩增可能会导致基因剂量的偏差。我们利用基于基因芯片的比较基因组杂交技术，对新鲜冷冻和福尔马林固定/石蜡包埋的肿瘤DNA进行了详细的研究。Pbi29从一对匹配的新鲜冷冻和福尔马林固定/石蜡包埋的肿瘤样本中扩增出DNA，具有相似的效率。在扩增的DNA中，基因剂量表示的失真是非随机的，并可重复性地涉及不同的基因组座位。区域扩增效率与模板基因组的区域GC含量显著相关。利用扩增的参比DNA可以有效地归一化扩增后的肿瘤DNA中有偏的基因表达。我们的数据表明，临床肿瘤样本中全基因组的基因剂量变化可以从数百个肿瘤细胞中可靠地评估出来。因此，这种扩增方法应该有助于对有限的肿瘤来源进行高通量的遗传分析，例如细针活检、激光显微解剖组织和小的石蜡包埋标本。
Sufficient quantity of genomic DNA can he a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Pbi29 amplified DNA from matched pairs of fresh-frozen and formalinfixed/parafrin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens.