A Direct Method to Isolate DNA from Phyllosphere Microbial Communities without Disrupting Leaf Tissues.

We developed a method for direct DNA isolation from phyllosphere microbial communities, designated Direct-DIP. This method comprises DNA extraction from non-shredded leaves with benzyl chloride, and DNA purification by gel filtration. Scanning electron microscopy showed that epiphytic microorganisms were completely removed from the leaf surface after benzyl chloride treatment, while microstructures of the leaf were not damaged. Clear DGGE profiles were obtained regardless of the plant species. Shannon diversity indices of DGGE profiles by Direct-DIP were higher than those by a conventional method. Our findings suggest that Direct-DIP is a rapid, simple, and cost-effective method of extracting DNA from phyllosphere microbial communities.

16S rRNA gene-based analysis of microbial community by whole-genome amplification and minigel-single-strand conformation polymorphism technique.

We have developed an analytical technique for the 16S rRNA gene that comprises whole-genome amplification and the polymerase chain reaction (PCR)-minigel-single-strand conformation polymorphism technique (WGA-SSCP). Under optimal conditions, SSCP bands could be detected when genomic DNA from bacteria of interest comprised 0.5% or more of the specimen. This method will be effective for the identification of nonculturable bacteria in a microbial community.

Molecular genetic diagnosis of familial tumors.

Recent advances in molecular biology have identified the responsible genes for many hereditary cancer syndromes (familial tumors). Therefore, now, the final diagnosis of familial tumors can be done by molecular methods to detect the genetic alteration in the disease-causing responsible gene(s) in the patient. So-called "genetic testing" is now available, not only as the diagnosis in the proband but also as the pre-symptomatic carrier diagnosis for unaffected family members. However, a number of issues, including legal, ethical, social, psychological, economic, and technical ones, surround and inhibit the clinical application of this testing, especially in Japan. As for molecular analysis to detect possible mutation in clinical specimens, highly sensitive scanning and detection methods are indispensable for screening nucleotide substitutions, including point mutations and single-nucleotide polymorphisms (SNPs). Also, several methods have been developed to screen and confirm the genetic alterations in familial tumors. These analytical techniques for detecting genetic alterations are categorized into four types, involving (1) scanning of nucleotide substitutions, (2) detection of defined mutations or SNPs, (3) size determination, and (4) DNA sequencing. Each technique has its advantages and disadvantages, and the technique in each case should be selected according to the purpose of the analysis. In this article, the concept of molecular diagnosis in familial tumors and the related molecular methods are described.