Borriello F, Weinberg D S, Mutter G L
Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115.
Diagn Mol Pathol. 1994 Dec;3(4):246-54. doi: 10.1097/00019606-199412000-00006.
In order to evaluate the feasibility of using quantitative polymerase chain reaction (PCR) to evaluate gene dosage, we developed an assay to detect alpha-globin genes, which are frequently deleted in alpha-thalassemia patients. In this quantitative assay alpha-1 and alpha-2 globin consensus regions are coamplified by one oligonucleotide pair, along with a second primer pair targeting a single-copy reference gene, namely, tumor necrosis factor alpha, or TNF-alpha. A series of DNA samples titrating alpha-globin against TNF-alpha DNA have a strong linear relationship between template ratios and product ratios (r > 0.98). Minimal sequence divergence (91% homology) between alpha-1 and alpha-2, internal to the identical primer annealing sites, results in a lower amplification efficiency for alpha-1, to 94% of alpha-2 for each cycle. Furthermore, when applied to a variety of individual DNA samples, the signal ratios of alpha-globin to TNF-alpha were far more variable than previously observed for titrated control DNA. We conclude that DNA isolates from different individuals may have idiosyncratic changes in amplification efficiency owing to polymorphic sequence variation and/or variable presence of unidentified contaminants. Despite these potentially confounding factors, however, we were able to identify by quantitative PCR a single gene deletion later confirmed by Southern blot analysis in 20 individual DNA samples.
为了评估使用定量聚合酶链反应（PCR）来评估基因剂量的可行性，我们开发了一种检测α-珠蛋白基因的方法，α-珠蛋白基因在α地中海贫血患者中经常缺失。在这种定量检测中，α1和α2珠蛋白共有区域由一对寡核苷酸共同扩增，同时另一对引物靶向一个单拷贝参考基因，即肿瘤坏死因子α（TNF-α）。一系列将α-珠蛋白与TNF-α DNA进行滴定的DNA样本，其模板比例与产物比例之间存在很强的线性关系（r>0.98）。α1和α2之间在相同引物退火位点内部的最小序列差异（91%同源性）导致α1的扩增效率较低，每个循环仅为α2的94%。此外，当应用于各种个体DNA样本时，α-珠蛋白与TNF-α的信号比例比之前滴定的对照DNA观察到的变化大得多。我们得出结论，由于多态性序列变异和/或未识别污染物的可变存在，来自不同个体的DNA分离物在扩增效率上可能存在特异变化。然而，尽管存在这些潜在的混杂因素，我们仍能够通过定量PCR鉴定出单个基因缺失，随后通过Southern印迹分析在20个个体DNA样本中得到证实。
