Department of Biology, University of Washington, Seattle, WA 98195-5325, USA.
Curr Genet. 2011 Aug;57(4):287-95. doi: 10.1007/s00294-011-0342-6. Epub 2011 May 4.
DNA sequences similar to those in the organellar genomes are also found in the nucleus. These non-coding sequences may be co-amplified by PCR with the authentic organellar DNA sequences, leading to erroneous conclusions. To avoid this problem, we describe an experimental procedure to prevent amplification of this "promiscuous" DNA when total tissue DNA is used with PCR. First, primers are designed for organelle-specific sequences using a bioinformatics method. These primers are then tested using methylation-sensitive PCR. The method is demonstrated for both end-point and real-time PCR with Zea mays, where most of the DNA sequences in the organellar genomes are also present in the nucleus. We use this procedure to quantify those nuclear DNA sequences that are near-perfect replicas of organellar DNA. This method should be useful for applications including phylogenetic analysis, organellar DNA quantification and clinical testing.
细胞核中也存在与细胞器基因组相似的 DNA 序列。这些非编码序列可能会与真实的细胞器 DNA 序列一起通过 PCR 被扩增，从而导致错误的结论。为了避免这个问题，我们描述了一种实验程序，用于在使用总组织 DNA 进行 PCR 时防止这种“混杂”DNA 的扩增。首先，使用生物信息学方法针对细胞器特异性序列设计引物。然后使用甲基化敏感 PCR 对这些引物进行测试。该方法在玉米的终点和实时 PCR 中都得到了验证，其中细胞器基因组中的大多数 DNA 序列也存在于细胞核中。我们使用该程序来定量那些与细胞器 DNA 近乎完美复制的核 DNA 序列。这种方法对于包括系统发育分析、细胞器 DNA 定量和临床检测在内的应用程序应该是有用的。
