Wallace A J, Wu C L, Elles R G
University Department of Medical Genetics, St. Mary's Hospital, Manchester, UK.
Genet Test. 1999;3(2):173-83. doi: 10.1089/gte.1999.3.173.
Many mutation scanning techniques are capable of locating mutations in DNA fragments much larger than the average exon. We have developed a system called Meta-PCR that can maximize the length of sequence scanned by these techniques, improving their productivity and realizing their full potential. Meta-PCR is a simple, versatile, and powerful method for generating chimeric DNA molecules. Currently, up to five PCR amplifiable fragments can be combined to form a single linear amplimer. The Meta-PCR reaction is self-assembling and takes place in two coupled stages carried out in a single reaction vessel. The order of fragments is reproducible and determined by primer design. We have developed two Meta-PCR assays, one comprising exons 6-10 of the Neurofibromatosis type 2 (NF2) gene and the second exons 8-12 of the human mismatch repair gene, hMLH1. We verified by direct sequencing that the order and sequence of the component exons in the Meta-PCR products is as predicted. Meta-PCR products from seven previously ascertained heterozygotes for NF2 mutations were directly sequenced. All seven mutations were clearly visible as mixed bases at the expected nucleotide, confirming that Meta-PCR faithfully reproduces the original sample genotype. We have evaluated the downstream use of the NF2 Meta-PCR products in fluorescent solid-phase chemical cleavage of mismatches (CCM). Meta-PCR products from eleven NF2 mutant heterozygotes were screened retrospectively for piperidine cleavage after hydroxylamine or potassium permanganate modification of mismatched bases. Ten of the 11 mutants were detected by visible cleavage. One mutation predicted to be cleaved after potassium permanganate modification was not detected. However, we were able to attribute this false negative to a failure in the CCM method. Meta-PCR is likely to be useful to clinical molecular diagnostic laboratories, helping them to fulfill demand for rapid and accurate screening for point mutations in large multi-exon genes.
许多突变扫描技术能够在比平均外显子大得多的DNA片段中定位突变。我们开发了一种称为Meta-PCR的系统，该系统可以最大限度地延长这些技术扫描的序列长度，提高其效率并充分发挥其潜力。Meta-PCR是一种简单、通用且强大的生成嵌合DNA分子的方法。目前，多达五个可通过PCR扩增的片段可以组合形成单个线性扩增子。Meta-PCR反应是自组装的，在单个反应容器中分两个耦合阶段进行。片段的顺序是可重复的，由引物设计决定。我们开发了两种Meta-PCR检测方法，一种包含2型神经纤维瘤病（NF2）基因的外显子6至10，另一种包含人类错配修复基因hMLH1的外显子8至12。我们通过直接测序验证了Meta-PCR产物中组成外显子的顺序和序列与预测一致。对七个先前确定的NF2突变杂合子的Meta-PCR产物进行了直接测序。所有七个突变在预期核苷酸处均清晰可见为混合碱基，证实Meta-PCR忠实地再现了原始样品的基因型。我们评估了NF2 Meta-PCR产物在荧光固相错配化学切割（CCM）中的下游应用。对11个NF2突变杂合子的Meta-PCR产物进行回顾性筛选，以检测错配碱基经羟胺或高锰酸钾修饰后哌啶切割的情况。11个突变体中有10个通过可见切割被检测到。一个预计在高锰酸钾修饰后会被切割的突变未被检测到。然而，我们能够将这种假阴性归因于CCM方法的失败。Meta-PCR可能对临床分子诊断实验室有用，帮助它们满足对大型多外显子基因中的点突变进行快速准确筛选的需求。
