PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 9933560

A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection.

B C Levin·H Cheng·D J Reeder
View original source ↗Export metadata ↓

Abstract

A human mitochondrial DNA (mtDNA) standard reference material (SRM 2392) will provide quality control when mtDNA is sequenced for forensic identifications, medical diagnosis, or mutation detection. SRM 2392 includes DNA from two lymphoblast cell cultures (CHR and 9947A) and cloned DNA from the CHR HV1 region, which contains a C stretch and is difficult to sequence. The mtDNA sequence (but not the DNA) of a third human template GM03798 is provided for comparison. Fifty-eight unique primer sets allow any area or the entire mtDNA (16,569 bp) to be amplified and sequenced. While none of the differences in these three templates correspond to published mutations associated with specific diseases, some of these differences did result in animo acid changes compared with that published by S. Anderson et al. (1981, Nature 290: 457-465). An interlaboratory evaluation of the amplification, sequencing, and data analysis of the CHR template was conducted by four laboratories. Corroboration of the SRM results will provide quality assurance that any unknown mtDNA is also being amplified and sequenced correctly.

Explore related subjects

Cell LineDNA Mutational AnalysisDNA PrimersDNA, MitochondrialForensic MedicineGene AmplificationHumansPolymerase Chain ReactionQuality ControlReference StandardsSequence Analysis, DNAJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

B C Levin, H Cheng, D J Reeder. 1999-01-15. A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection.. https://doi.org/10.1006/geno.1998.5513

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2019

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis.

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

Cell Line↗
PubMed2015

NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability.

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

Cell Line↗
PubMed2014

Upregulation of TLR2 expression is induced by estrogen via an estrogen-response element (ERE).

TLR2 and estrogen are both thought to be involved in the pathogenesis of RA; however, it is unknown if there is an association between estrogen and TLR2. In this report, we treated PMA-differentiated THP-1 cells with 17β-estradiol (E2) and observed increases in TLR2 mRNA and protein levels by real-time quantitative PCR and western blot. Transfection of THP-1 cells with a series of 5'-deleted TLR2 promoter-luciferase constructs revealed that E2 enhanced TLR2 transcriptional activity in an estrogen receptor alpha (ERα)-dependent pattern. An estrogen receptor response element (ERE) was identified 251 bases upstream of the TLR2 promoter, and electrophoretic mobility shift assay and chromatin immunoprecipitations showed ERα binding was increased by E2. In summary, this work demonstrated that TLR2 is a new estrogen-regulated gene whose expression is upregulated through the interaction of ERα with an ERE in the promoter region.

Cell Line↗