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.

Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Abstract: Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan® assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan®probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRASgene in the presence of a 200 000-fold excess of unmutated KRASgenes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan®probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAScodon 12 in parallel in a single experiment.

Pack-MULE transposable elements mediate gene evolution in plants

Abstract: Mutator-like transposable elements (MULEs) are found in many eukaryotic genomes and are especially prevalent in higher plants. In maize, rice and Arabidopsis a few MULEs were shown to carry fragments of cellular genes. These chimaeric elements are called Pack-MULEs in this study. The abundance of MULEs in rice and the availability of most of the genome sequence permitted a systematic analysis of the prevalence and nature of Pack-MULEs in an entire genome. Here we report that there are over 3,000 Pack-MULEs in rice containing fragments derived from more than 1,000 cellular genes. Pack-MULEs frequently contain fragments from multiple chromosomal loci that are fused to form new open reading frames, some of which are expressed as chimaeric transcripts. About 5% of the Pack-MULEs are represented in collections of complementary DNA. Functional analysis of amino acid sequences and proteomic data indicate that some captured gene fragments might be functional. Comparison of the cellular genes and Pack-MULE counterparts indicates that fragments of genomic DNA have been captured, rearranged and amplified over millions of years. Given the abundance of Pack-MULEs in rice and the widespread occurrence of MULEs in all characterized plant genomes, gene fragment acquisition by Pack-MULEs might represent an important new mechanism for the evolution of genes in higher plants.

The Genome of the African Trypanosome

Abstract: Trypanosoma brucei is a diploid organism with a nuclear haploid DNA content of 35 ± 9 megabase pairs (Mb) depending on the trypanosome isolate. About 15% of the total cellular DNA is in the kinetoplast where it is organized as homogenous 23-kb maxicircles and heterogeneous 1 -kb minicircles. The remaining 85% of the DNA occurs in the nucleus as linear DNA molecules ranging in size from 50 kb to 6 Mb. At least 11 pairs of megabase chromosomes of 1 to 6 Mb exist that are numbered I-XI from smallest to largest. The two homologues of a megabase chromosome pair can differ in size by as much as 4-fold. Several intermediate-sized chromosomes of 0.2 – 0.9 Mb and uncertain ploidy are also present. The telomeres of the megabase and intermediate chromosomes are linked to expression sites for the genes encoding the variant surface glycoproteins (VSGs). In addition, about 100 linear minichromosomes of 50–150 kb occur and serve as repositories for unexpressed, telomere-linked VSG genes. About 50% of the nuclear genome is coding sequence. To date only one tRNA gene and one protein-encoding gene, specifying poly(A) polymerase, have been found to contain an intron. The complete sequence determination of chromosomes I and II (about 1 Mb each) is nearing completion and more than 20 Mb of discontinuous single-pass genomic DNA sequence data have been generated. Based on analogy with the Leishmania genome, much of the African trypanosome nuclear genome is likely to be arrayed as long transcription units of 50 or more intronless genes. Knowledge of this genomic sequence and its complete set of genes will open many new avenues for identifying better ways to control or eliminate this pathogen and its deadly disease.