Department of Anaesthesiology and Intensive Care Medicine, University Hospital Bonn, Bonn, Germany.
Biotechniques. 2009 Dec;47(6):1023-8. doi: 10.2144/000113300.
The reliable quantification of gene copy number variations is a precondition for future investigations regarding their functional relevance. To date, there is no generally accepted gold standard method for copy number quantification, and methods in current use have given inconsistent results in selected cohorts. In this study, we compare two methods for copy number quantification. beta-defensin gene copy numbers were determined in parallel in 80 genomic DNA samples by real-time PCR and multiplex ligation-dependent probe amplification (MLPA). The pyrosequencing-based paralog ratio test (PPRT) was used as a standard of comparison in 79 out of 80 samples. Realtime PCR and MPLA results confirmed concordant DEFB4, DEFB103A, and DEFB104A copy numbers within samples. These two methods showed identical results in 32 out of 80 samples; 29 of these 32 samples comprised four or fewer copies. The coefficient of variation of MLPA is lower compared with PCR. In addition, the consistency between MLPA and PPRT is higher than either PCR/MLPA or PCR/PPRT consistency. In summary, these results suggest that MLPA is superior to real-time PCR in beta-defensin copy number quantification.
可靠的基因拷贝数变异定量是未来研究其功能相关性的前提条件。迄今为止，尚无被普遍接受的拷贝数定量金标准方法，并且目前使用的方法在选定的队列中给出了不一致的结果。在这项研究中，我们比较了两种拷贝数定量方法。通过实时 PCR 和多重连接依赖性探针扩增（MLPA），同时在 80 个基因组 DNA 样本中确定了β-防御素基因拷贝数。基于焦磷酸测序的同源基因比例测试（PPRT）在 80 个样本中的 79 个作为比较标准。实时 PCR 和 MPLA 结果证实了样本内 DEFB4、DEFB103A 和 DEFB104A 拷贝数的一致性。这两种方法在 80 个样本中的 32 个中显示出相同的结果；这 32 个样本中有 29 个样本包含 4 个或更少的拷贝。MLPA 的变异系数低于 PCR。此外，MLPA 与 PPRT 的一致性高于 PCR/MLPA 或 PCR/PPRT 的一致性。总之，这些结果表明 MLPA 在β-防御素拷贝数定量方面优于实时 PCR。
