Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.

Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers.

Comparing binding site information to binding affinity reveals that Crp/DNA complexes have several distinct binding conformers.

We show that the cAMP receptor protein (Crp) binds to DNA as several different conformers. This situation has precluded discovering a high correlation between any sequence property and binding affinity for proteins that bend DNA. Experimentally quantified affinities of Synechocystis sp.

Algorithm for writing a scientific manuscript.

We present an algorithm for the construction of a strong initial draft. It is designed to overcome writer's block and to assist scientists who are not native English speakers. The writing starts with making figures and tables.

Novel means of viral antigen identification: improved detection of avian influenza viruses by proximity ligation.

Recent outbreaks of avian influenza in different parts of the world have caused major economic losses for the poultry industry, affected wildlife seriously and present a significant threat even to human public health, due to the risk for zoonotic transmission. The ability to recognize avian influenza viruses (AIVs) early is of paramount importance to ensure that appropriate measures can be taken quickly to contain the outbreak. In this study, the performance of a proximity ligation assay (PLA) for the detection of AIV antigens in biological specimens was evaluated.

Chromosome organization and chromatin modification: influence on genome function and evolution.

Histone modifications of nucleosomes distinguish euchromatic from heterochromatic chromatin states, distinguish gene regulation in eukaryotes from that of prokaryotes, and appear to allow eukaryotes to focus recombination events on regions of highest gene concentrations. Four additional epigenetic mechanisms that regulate commitment of cell lineages to their differentiated states are involved in the inheritance of differentiated states, e.g.

Detection of bovine viral diarrhoea virus infected cattle--testing tissue samples derived from ear tagging using an Erns capture ELISA.

A new diagnostic approach testing tissue samples derived from cattle ear tagging for bovine viral diarrhoea virus (BVDV) antigen in a commercially available antigen capture enzyme-linked immunosorbent assay (ACE) was developed. To validate this method, 99 positive and 469 negative samples were tested. With those samples the assay yielded a sensitivity of 100% and specificity of >or=99.

Repositioning-dependent fate of duplicate genes.

Gene duplication is the main source of evolutionary novelties. However, the problem with duplicates is that the purifying selection overlooks deleterious mutations in the redundant sequence, which therefore, instead of gaining a new function, often degrades into a functionless pseudogene. This risk of functional loss instead of gain is much higher for small populations of higher organisms with a slow and complex development.

Repair rates of R-band, G-band and C-band DNA in murine and human cultured cells.

Repair of cyclobutane pyrimidine dimers (CPDs) in cultured neonatal human fibroblasts and in Mus spretus x M. castaneus F1 neonatal skin fibroblasts was analyzed after UVC-irradiation by cleavage with T4 endonuclease V cyclopyrimidine dimer glycosylase, alkaline-agarose gel electrophoresis, and Southern blotting. The blots were sequentially probed with 32P-labeled Alu, or B2, to preferentially illuminate R-band DNA, by L1 to preferentially illuminate G-band DNA, and by satellite DNA to illuminate C-band DNA.

Cancerous hyper-mutagenesis in p53 genes is possibly associated with transcriptional bypass of DNA lesions.

The database of tumor-associated p53 base substitutions includes about 5% of tumors with two or more base substitutions. These multiplet base substitutions in one tumor are evidence for hyper-mutagenesis. Our retrospective analysis of this database indicates that most multiplets arise from a single transient hyper-mutagenic event in one cell that subsequently proliferated into a clonal tumor.

Cell-selfish modes of evolution and mutations directed after transcriptional bypass.

During transcription, prokaryotic and eukaryotic RNA polymerases bypass and misread (transcriptional mutagenesis) several classes of DNA lesions. For example, misreading of 8-OH-dG generates mRNAs containing G to T transversions. After translation, if the mutant protein briefly allowed the cell a growth-DNA replication advantage, then precocious DNA replication would bypass that unrepaired 8-OH-dG and misinsert dA opposite the directing DNA lesion with a higher probability than would be experienced for 8-OH-G lesions at other positions in otherwise identical neighboring cells.

Ligation-mediated PCR: robotic liquid handling for DNA damage and repair.

The investigation of in vivo DNA repair in mammalian cells at nucleotide resolution requires the quantification of break frequencies less than one per kilobase. By optimizing several parameters of the ligation-mediated PCR technique, we find that the required sensitivity can be achieved. We also report details of a one-day procedure that can be performed either with or without a robotic liquid handling workstation.

High-efficiency DNA ligation for clamp attachment without polymerase chain reaction.

We coupled ligation with mass action to achieve high-efficiency clamp attachment without polymerase chain reaction (PCR). Using a 10-fold molar excess of a GC-rich clamp of synthesized and hybridized oligonucleotides, we achieved the maximum clamp-ligation efficiency in which the clamp was ligated to >95% of 10(10)-10(12) restriction ends of a PCR-amplified fragment. The maximum efficiency was confirmed by ligating the clamp to 10(11)-10(12) restriction ends of human genomic DNA.

Evidence for DNA charge transport in the nucleus.

Oxidative damage to DNA bases in isolated HeLa nuclei occurs upon treatment with rhodium intercalators and photoactivation. Oxidation occurs preferentially at the 5'-guanine of 5'-GG-3' sites, indicative of base damage by DNA-mediated charge transfer chemistry. Moreover, oxidative damage occurs at protein-bound sites which are inaccessible to rhodium.

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing.

We describe a fluorescence-based directed termination PCR (fluorescent DT-PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer.

Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology.

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR).

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR.

Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair. In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy. We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer.

Mapping of peroxyl radical induced damage on genomic DNA.

We have examined the DNA damage produced by reaction of peroxyl radicals with human fibroblast DNA. DNA damage consisted of both strand breaks and base modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitation of fragment size distributions using denaturing glyoxal-agarose gel electrophoresis.

Adaptive enhancement and kinetics of nucleotide excision repair in humans.

An adaptive response, low doses of a mutagen rendering cells more able to subsequently cope with higher doses of that or a related challenging mutagen, enhances nucleotide excision repair in human fibroblasts. After fibroblasts were flashed with 20 J/m2 of UVC, the cyclopyrimidine dimer frequency at any single dinucleotide position remained unchanged for several hours before abruptly displaying first order kinetics of repair. These kinetics were determined by ligation-mediated PCR along exon 9 of the human p53 gene.

CpG transition strand asymmetry and hitch-hiking mutations as measures of tumorigenic selection in shaping the p53 mutation spectrum.

By the genetic code, the average protein perturbation expected from a CpG-->TpG transition is strand-specific and smallest when it originates with the C on the transcribed (noncoding) strand. To distinguish the effects of selection from mutagenesis, we measured strand asymmetry for CpG-->TpG transitions fixed in active p53 genes and pseudogenes during vertebrate evolution, and for p53 genes from human tumors with one (singlet) and two (doublet) p53 point mutations. Mutagenesis appears to generate the transitions symmetrically while selection usually acts asymmetrically being most sensitive to the larger protein perturbations.

Heterogeneous repair of N-methylpurines at the nucleotide level in normal human cells.

Base excision repair rates of dimethyl sulfate-induced 3-methyladenine and 7-methylguanine adducts were measured at nucleotide resolution along the PGK1 gene in normal human fibroblasts. Rates of 7-methylguanine repair showed a 30-fold dependence on nucleotide position, while position-dependent repair rates of 3-methyladenine varied only sixfold. Slow excision rates for 7-methylguanine bases afforded the opportunity to study their excision in vitro as a model for base excision repair.

Chronic low-dose lesion equilibrium along genes: measurement, molecular epidemiology, and theory of the minimal relevant dose.

Spontaneous mutations seem to be caused almost entirely by endogenous lesions. The pattern of these lesions along a gene represents an equilibrium between damage and repair. A pattern can be measured using ligation-mediated PCR (polymerase chain reaction) and a large chronic dose of a suspected endogenous mutagen.

Endogenous lesions, S-phase-independent spontaneous mutations, and evolutionary strategies for base excision repair.

We calculate from published levels of endogenous base lesions that our cells constantly generate and excise during base excision repair (BER) about one million lesions per day. Repair glycosylases may also non-specifically excise an additional number of undamaged bases. The resulting abasic sites are repaired daily by BER.

Mobile genetic elements, chiasmata, and the unique organization of beta-heterochromatin.

Beta-heterochromatin in Drosophila and the Syrian hamster share a similar DNA organization, few unique sequences, and scrambled repeats of mobile elements without tandem repetition. DNA in alpha-heterochromatin is tandemly repetitious, and we now show that the repeat unit can either contain or lack a mobile element. The tandem repeat organization of alpha-heterochromatin is presumably due to a concertina-like mechanism of unequal exchange between repeat units.