Nuclear DNA

About: Nuclear DNA is a research topic. Over the lifetime, 3933 publications have been published within this topic receiving 185830 citations.

DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR.

Abstract: Forensic laboratories are increasingly confronted with problematic samples from the scene of crime, containing only minute amounts of deoxyribonucleic acid (DNA), which may include polymerase chain reaction (PCR)-inhibiting substances. Efficient DNA extraction procedures, as well as accurate DNA quantification methods, are critical steps involved in the process of successful DNA analysis of such samples. The phenol-chloroform method is a sensitive method for the extraction of DNA from a wide variety of forensic samples, although it is known to be laborious compared with single-tube extraction methods. The relatively high DNA recovery and the quality of the extracted DNA speak for itself. For reliable and sensitive DNA quantitation, the application of real-time PCR is described. We modified a published real-time PCR assay, which allows for the combined analysis of nuclear and mitochondrial DNA, by introducing 1) improved hybridization probes with the use of minor groove binders; 2) an internal positive control (for both nuclear and mitochondrial DNA) for the detection of PCR inhibitors; and 3) different amplicon lengths for the determination of the degradation state of the DNA. The internal positive controls were constructed by site directed mutagenesis by overlap extension of the wild-type mitochondrial and nuclear DNA target with the advantage that no additional probes, which are cost-intensive, are required. The quantitation system is accomplished as a modular concept, which allows for the combined determination of the above-mentioned features (quantity/inhibition or quantity/degradation) depending on the situation.

Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs

Abstract: Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.

Length mutations in human mitochondrial dna

Abstract: By high-resolution, restriction mapping of mitochondrial DNAs purified from 112 human individuals, we have identified 14 length variants caused by small additions and deletions (from about 6 to 14 base pairs in length). Three of the 14 length differences are due to mutations at two locations within the D loop, whereas the remaining 11 occur at seven sites that are probably within other noncoding sequences and at junctions between coding sequences. In five of the nine regions of length polymorphism, there is a sequence of five cytosines in a row, this sequence being comparatively rare in coding DNA. Phylogenetic analysis indicates that, in most of the polymorphic regions, a given length mutation has arisen several times independently in different human lineages. The average rate at which length mutations have been arising and surviving in the human species is estimated to be many times higher for noncoding mtDNA than for noncoding nuclear DNA. The mystery of why vertebrate mtDNA is more prone than nuclear DNA to evolve by point mutation is now compounded by the discovery of a similar bias toward rapid evolution by length mutation.

Novel expression-linked copies of the genes for variant surface antigens in trypanosomes.

Abstract: Pathogenic African trypanosomes evade the immune system of their mammalian hosts by the sequential expression of alternative cell-surface glycoproteins (reviewed in refs 1,2). Variant surface glycoproteins (VSGs) purified from cloned variants of Trypanosoma brucei have similar molecular weights (about 60,000), but differ in amino acid composition, N-terminal amino acid sequence and C-terminal structure. We have cloned DNA complementary to the messenger RNA's for four immunologically distinct VSGs and hybridised these complementary DNAs (cDNAs) with restriction digests of T. brucei nuclear DNA, fractionated by gel electrophoresis and transferred to nitrocellulose strips. Each cDNA recognises a unique set of fragments and this basic set is present unaltered in the nuclear DNAs from the four variants. In addition, each probe recognises an extra fragment only in nuclear DNA isolated from cells expressing the VSG corresponding to the cDNA probe. We infer that activation of a VSG gene involves the production of an expression-linked copy of that gene.