genomic DNA

About: genomic DNA is a research topic. Over the lifetime, 15046 publications have been published within this topic receiving 663636 citations. The topic is also known as: genomic deoxyribonucleic acid & gDNA.

Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors

Abstract: Genomic DNA copy number alterations are key genetic events in the development and progression of human cancers. Here we report a genome-wide microarray comparative genomic hybridization (array CGH) analysis of DNA copy number variation in a series of primary human breast tumors. We have profiled DNA copy number alteration across 6,691 mapped human genes, in 44 predominantly advanced, primary breast tumors and 10 breast cancer cell lines. While the overall patterns of DNA amplification and deletion corroborate previous cytogenetic studies, the high-resolution (gene-by-gene) mapping of amplicon boundaries and the quantitative analysis of amplicon shape provide significant improvement in the localization of candidate oncogenes. Parallel microarray measurements of mRNA levels reveal the remarkable degree to which variation in gene copy number contributes to variation in gene expression in tumor cells. Specifically, we find that 62% of highly amplified genes show moderately or highly elevated expression, that DNA copy number influences gene expression across a wide range of DNA copy number alterations (deletion, low-, mid- and high-level amplification), that on average, a 2-fold change in DNA copy number is associated with a corresponding 1.5-fold change in mRNA levels, and that overall, at least 12% of all the variation in gene expression among the breast tumors is directly attributable to underlying variation in gene copy number. These findings provide evidence that widespread DNA copy number alteration can lead directly to global deregulation of gene expression, which may contribute to the development or progression of cancer.

Resolving individuals contributing trace amounts of DNA to highly complex mixtures using high-density SNP genotyping microarrays.

Abstract: We use high-density single nucleotide polymorphism (SNP) genotyping microarrays to demonstrate the ability to accurately and robustly determine whether individuals are in a complex genomic DNA mixture. We first develop a theoretical framework for detecting an individual's presence within a mixture, then show, through simulations, the limits associated with our method, and finally demonstrate experimentally the identification of the presence of genomic DNA of specific individuals within a series of highly complex genomic mixtures, including mixtures where an individual contributes less than 0.1% of the total genomic DNA. These findings shift the perceived utility of SNPs for identifying individual trace contributors within a forensics mixture, and suggest future research efforts into assessing the viability of previously sub-optimal DNA sources due to sample contamination. These findings also suggest that composite statistics across cohorts, such as allele frequency or genotype counts, do not mask identity within genome-wide association studies. The implications of these findings are discussed.

Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms.

Abstract: Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G1C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation. The biosphere is dominated by microorganisms (32), yet most microbes in nature have not been studied. Traditional methods for culturing microorganisms limit analysis to those that grow under laboratory conditions (14, 25). The recent surge of research in molecular microbial ecology provides compelling evidence for the existence of many novel types of microorganisms in the environment in numbers and varieties that dwarf those of the comparatively few microorganisms amenable to laboratory cultivation (7, 13, 31). Corroboration comes from estimates of DNA complexity and the discovery of many unique 16S rRNA gene sequences from numerous environmental sources (8, 10, 28). Collectively, the genomes of the total microbiota found in nature, which we termed the metagenome (11), contain vastly more genetic information than is contained in the culturable subset. Given the profound utility and importance of microorganisms to all biological systems, methods are needed to access the wealth of information within the metagenome. Cloning large fragments of DNA isolated directly from microbes in natural environments provides a method to access soil metagenomic DNA. Previously, we investigated the use of the bacterial artificial chromosome (BAC) vector to express Bacillus cereus genomic DNA (20). The advantage of BAC vectors is that they maintain very large DNA inserts (greater than 100 kb) stably in Escherichia coli (23), facilitating the cloning of large fragments of DNA. Our results demonstrated that expression of heterologous DNA from B. cereus in an E. coli BAC system was detectable at a reasonable frequency (20), validating the idea that the low-copy BAC vector (one to two per cell) (23) could be used to express foreign DNA from foreign promoters in E. coli. Here we describe the construction and initial screening of two BAC libraries made with DNA isolated directly from soil. We found detectable levels of several biochemical activities from BAC library clones. Sequence analysis of selected BAC plasmids encoding such activities and of 16S rRNA genes in one of the libraries confirms the novelty of the genomic information cloned in our libraries. The results show that DNA extracted directly from soil is a valuable source of new genetic information and is accessible by using BAC libraries. Our results demonstrate that both traditional and functional genomics of uncultured microorganisms can be carried out by this approach and that screening of metagenome libraries for activities or gene sequences can provide a basis for conducting genomic analyses of uncultured microorganisms.