Gene assembling: a new approach in molecular diagnosis of hereditary breast cancer

Many disease susceptibility genes are large and consist of many exons in which point mutations are scattered throughout. Scanning each exon individuallyrepresents a tedious task which can be time consuming and expensive. There hasbeen increasing demand for rapid and accurate methods for full scanning of unknownpoint mutations in large multi-exon genes. Gene Assembling is a new method for creatingchimeric DNA molecules using a modified PCR reaction that allows maximizingthe length of sequence that can be scanned by sensitive downstream technique. In the present study assembling of exons 2, 20, 23 and 24 of the BRCA1 gene and their subsequent analysis by direct sequencing is demonstrated. The BRCA1 exons 2 and 20 are hot spot regions that are known to harbor particularly deleterious mutations. In order to avoid missing any mutation in these two exons, the four exons previously mentioned were assembled in the following order of preferences: 23, 20, 2 and 24. However, the order of fragments can be predetermined by primer design. The order and sequence of the component exons in the gene-assembled products were characterized by direct sequencing as predicted. Gene-assembled products from three previously ascertained heterozygotes for BRCA1 mutations were directly sequenced and gave the same sequence patterns. This experience suggests that Gene Assembling technique could be applied as a highly sensitive and cost-effective method in identifying mutation in complexgenes such as BRCA and ColA1/2 helping clinical molecular diagnostic laboratories,to fulfill the demand for scanning complex genetic diseases at a lower cost.