Genetic and expression analysis of SNPs in the human deoxyribonuclease II: SNPs in the promoter region reduce its in vivo activity through decreased promoter activity

Five SNPs in the human DNase II gene have been reported to be associated with rheumatoid arthritis (RA). Genotype and haplotype analysis of 14 SNPs, nine SNPs of which reported in the NCBI dbSNP database in addition to these five SNPs, was performed in healthy subjects. The enzymatic activities of the amino acid substituted DNase II corresponding to each SNP and serum DNase II in healthy Japanese, and promoter activities derived from each haplotype of the RA‐related SNPs were measured. Significant correlations between genotype in each RA‐related SNP and enzymatic activity levels were found; alleles associated with RA exhibited a reduction in serum DNase II activity. Furthermore, the promoter activities of each reporter construct corresponding to predominant haplotypes in three SNPs in the promoter region of the gene exhibited significant correlation with levels of serum DNase II activity. These findings indicate these three SNPs could alter the promoter activity of DNASE2, leading to a decline in DNase II activity in the serum through gene expression. Since the three SNPs in the promoter region of the DNase II gene could affect in vivo DNase II activity through reduction of the promoter activity, it is feasible to identify these SNPs susceptible to RA.     

Electrochemical SNPs detection using an abasic site-containing DNA on a gold electrode

An abasic site-containing DNA combined with lumiflavin allows amperometric determination of single nucleotide polymorphism through hydrogen bond-mediated nucleobase recognition in water by using abasic sites as a molecular recognition field.     

Exploring of tri-allelic SNPs using pyrosequencing and the SNaPshot methods for forensic application

Tri-allelic single nucleotide polymorphisms (SNPs) are potential forensic markers for DNA analysis. Currently, only a limited number of tri-allelic SNP loci have been proved to be fit for forensic application. In this study, we aimed to develop an effective method to select and genotype tri-allelic SNPs based on both Pyrosequencing (PSQ) and the SNaPshot methods. 50 candidate SNPs were chosen from NCBI's dbSNP database and were analyzed by PSQ. The results revealed that 20 SNPs were tri-allelic and were located on 16 autosomal chromosomes. Then 20 SNP loci were combined in one multiplex polymerase chain reaction to develop a single base extension (SBE)-based SNP-typing assay. A total of 100 unrelated Chinese individuals were genotyped by this assay and allele frequencies were estimated. The total discrimination power was 0.999999999975 and the cumulative probability of exclusion was 0.9937. These data demonstrated that the strategy is a rapid and effective method for seeking and typing tri-allelic SNPs. In addition, the 20 tri-allelic SNP multiplex typing assay may be used to supplement paternity testing and human identification.     

East Asian mtDNA haplogroup determination in Koreans: haplogroup-level coding region SNP analysis and subhaplogroup-level control region sequence analysis

The present study analyzed 21 coding region SNP markers and one deletion motif for the determination of East Asian mitochondrial DNA (mtDNA) haplogroups by designing three multiplex systems which apply single base extension methods. Using two multiplex systems, all 593 Korean mtDNAs were allocated into 15 haplogroups: M, D, D4, D5, G, M7, M8, M9, M10, M11, R, R9, B, A, and N9. As the D4 haplotypes occurred most frequently in Koreans, the third multiplex system was used to further define D4 subhaplogroups: D4a, D4b, D4e, D4g, D4h, and D4j. This method allowed the complementation of coding region information with control region mutation motifs and the resultant findings also suggest reliable control region mutation motifs for the assignment of East Asian mtDNA haplogroups. These three multiplex systems produce good results in degraded samples as they contain small PCR products (101-154 bp) for single base extension reactions. SNP scoring was performed in 101 old skeletal remains using these three systems to prove their utility in degraded samples. The sequence analysis of mtDNA control region with high incidence of haplogroup-specific mutations and the selective scoring of highly informative coding region SNPs using the three multiplex systems are useful tools for most applications involving East Asian mtDNA haplogroup determination and haplogroup-directed stringent quality control.     

Nanoparticle coding: size-based assays using atomic force microscopy

Described herein is a novel strategy for the construction and interrogation of an assay platform based on (1) the size encoding of labeled nanoparticles; (2) the high imaging resolution of atomic force microscopy; and (3) evaporatively driven self-assembly of dense nanoparticle layers. This strategy employs two different sized nanoparticles that couple in the presence of a target analyte. In this example, one set of particles is a few hundred nanometers in size and acts as a capture substrate, while a second set of smaller particles serve as the analyte label. Thus, by forming an evaporatively assembled layer from a mixture of the two particle dispersions, the imaged size of the smaller particles when bound to the larger capture particles identifies the presence of the analyte. This letter demonstrates the feasibility of our bar-code strategy by concept tests using the binding specificity of biotin-modified silica nanoparticles (300-nm diameter) with streptavidin-labeled gold nanoparticles (10-nm diameter). The potential to extensively multiplex this assay strategy is briefly discussed.     

Dissection of molecular and histological subtypes of papillary thyroid cancer using alternative splicing profiles

Despite growing evidence of the relevance of alternative splicing (AS) to cancer development and progression, the biological implications of AS for tumor behaviors, including papillary thyroid cancer (PTC), remain elusive. With the aim of further understanding the molecular and histological subtypes of PTC, we in this study explored whether AS events might act as new molecular determinants. For this purpose, AS profiles were analyzed in RNA-sequencing data from The Cancer Genome Atlas (TCGA) and from a Korean patient dataset. A total of 23 distinct exon-skipping (ES) events that correlated significantly with PTC oncogenic activity and differentiation scores were identified. The two top-ranked ES events, NUMA1_17515 in exon 18 of NUMA1 and TUBB3_38175 in exon 6 of TUBB3, showed high correlations with oncogenic activities and discriminated histological and molecular subtypes of PTC. Furthermore, two novel intron-retention (IR) events for TUBB3 were uncovered. All ES and IR events for the TUBB3 gene were predicted to induce nonsense-mediated mRNA decay. The relative abundances of intron reads in the PTC dataset from TCGA showed IR levels to differ significantly among PTC subtypes, possibly reflecting their different tumor behaviors. This study provides a landscape of AS changes among PTC subtypes and identified two significant AS events, NUMA1_17515 and TUBB3_38175, as potential AS biomarkers for PTC subclassification and characterization. The AS events identified in this study may be involved in the development of phenotypic differences underlying the functional characteristics and histological differentiation of PTCs.Subject terms: Cancer genomics, RNA splicing     

A digital coding combination analysis for mutational genotyping using pyrosequencing

In the present study, we developed a novel digital coding combination analysis (DCCA) to analyze the gene mutation based on the sample combination principle. The principle is that any numerically named sample is divided into two groups, any two samples are not grouped in the same two groups, and any sample can be tested within the detection limit. Therefore, we proposed a specific combination that N samples were divided into M groups. Then N samples were analyzed, which could obtain the mutation results of M mixed groups. If only two groups showed positive (mutant type) signals, the same sample number from two positive signal groups would be the positive sample, and the remaining samples were negative (wild type). If three groups or more exhibited positive results, the same sample number from three positive signal groups would be the positive sample. If some samples remained uncertain, individual samples could be analyzed on a small scale. In the present study, we used the two genotypes of a mutation site (A5301G) to verify whether it was a useful and promising method. The results showed that we could quantitatively detect mutations and demonstrate 100% consistent results against a panel of defined mixtures with the detection limit using pyrosequencing. This method was suitable, sensitive, and reproducible for screening and analyzing low-frequency mutation samples, which could reduce reagent consumption and cost by approximately 70–80% compared with conventional clinical methods.     

High-throughput profiling of the mitochondrial proteome using affinity fractionation and automation

Recent studies have demonstrated the need for complementing cellular genomic information with specific information on expressed proteins, or proteomics, since the correlation between the two is poor. Typically, proteomic information is gathered by analyzing samples on two-dimensional gels with the subsequent identification of specific proteins of interest by using trypsin digestion and mass spectrometry in a process termed peptide mass fingerprinting. These procedures have, as a rule, been labor-intensive and manual, and therefore of low throughput. The development of automated proteomic technology for processing large numbers of samples simultaneously has made the concept of profiling entire proteomes feasible at last. In this study, we report the initiation of the (eventual) complete profile of the rat mitochondrial proteome by using high-throughput automated equipment in combination with a novel fractionation technique using minispin affinity columns. Using these technologies, approximately one hundred proteins could be identified in several days. In addition, separate profiles of calcium binding proteins, glycoproteins, and hydrophobic or membrane proteins could be generated. Because mitochondrial dysfunction has been implicated in numerous diseases, such as cancer, Alzheimer's disease and diabetes, it is probable that the identification of the majority of mitochondrial proteins will be a beneficial tool for developing drug and diagnostic targets for associated diseases.     

Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis

Mitochondrial disorders are a group of clinically and genetically heterogeneous diseases. Common recurrent mitochondrial DNA (mtDNA) point mutations account for the molecular defects of a small proportion of patients. In order to identify mtDNA mutations, comprehensive mutational analysis of the entire mitochondrial genome is necessary. We developed the temporal temperature gradient gel electrophoresis (TTGE) method to screen for mutations in mtDNA. The entire mitochondrial genome was amplified using 32 pairs of overlapping primers followed by TTGE analysis of the DNA fragments. TTGE method was first validated on 200 DNA fragments containing known mutations or polymorphisms. On TTGE, homoplasmic nucleotide substitutions show a single band shift and heteroplasmic mutations show multiple banding patterns. The known mutations or polymorphisms were correctly identified. TTGE was then used to screen for unknown mutations in the mitochondrial genome. DNA banding patterns, deviated from wild-type, suggestive of either homoplasmic or heteroplasmic mutations, were followed by direct DNA sequencing to identify the mutations. Numerous mutations and polymorphisms were detected. The results demonstrated that TTGE detects and distinguishes heteroplasmic mutations from homoplasmic polymorphisms. It also detects heteroplasmic changes in the background of a homoplasmic polymorphism. Overall, TTGE was proven to be a simple, rapid, sensitive, and effective mutation detection method.     

Dissection of complex molecular recognition interfaces

The synthesis of a family of zinc porphyrins and pyridine ligands equipped with peripheral H-bonding functionality has provided access to a wide range of closely related supramolecular complexes featuring between zero and four intramolecular H-bonds. An automated UV/vis titration system was used to characterize 120 different complexes, and these data were used to construct a large of number of different chemical double mutant cycles to quantify the intramolecular H-bonding interactions. The results probe the quantitative structure-activity relationship that governs cooperativity in the assembly of complex molecular recognition interfaces. Specifically, variations in the chemical structures of the complexes have allowed us to change the supramolecular architecture, conformational flexibility, geometric complementarity, the number and nature of the H-bond interactions, and the overall stability of the complex. The free energy contributions from individual H-bonds are additive, and there is remarkably little variation with architecture in the effective molarity for the formation of intramolecular interactions. Intramolecular H-bonds are not observed in complexes where they are geometrically impossible, but there are no cases where excellent geometric complementarity leads to very high affinities. Similarly, changes in conformational flexibility seem to have limited impact on the values of effective molarity (EM). The major variation that was found for all of the 48 intramolecular interactions that were examined using double mutant cycles is that the values of EM for intramolecular carboxylate ester-phenol H-bonds (200 mM) are an order of magnitude larger than those found for phosphonate diester-phenol H-bonds (30 mM). The corresponding intermolecular phosphonate diester-phenol H-bonds are 2 orders of magnitude more stable than carboxylate ester-phenol H-bonds, and the large differences in EM may be due to some kind of compensation effect, where the stronger H-bond is harder to make, because it imposes tighter constraints on the geometry of the complex.     

Studies of end-groups in polystyrene using H NMR

Studies have been made by 400 MHz ¹H NMR of initiator fragments in polystyrene made by radical polymerization in solution at 60 °C. Azoisobutyronitrile, benzoyl peroxide and lauroyl peroxide have been used as initiators. The peaks arising from hydrogens in the end-groups have been recognized. They cause alterations in the ratio of “aromatic hydrogens” to “aliphatic hydrogens” for polystyrene.     

Evidence for the optical signalling of changes in bicarbonate concentration within the mitochondrial region of living cells

Image and spectral intensity from bicarbonate-selective europium(III) probes localised in the mitochondria of cells is modulated reversibly by variation of external pCO(2), and is suppressed by addition of the carbonic anhydrase inhibitor, acetazolomide.     

Genotyping fetal paternally inherited SNPs by MALDI-TOF MS using cell-free fetal DNA in maternal plasma: influence of size fractionation

The determination of fetal point mutations from fetal cell-free DNA (cf-DNA) in maternal plasma is technically challenging due to the preponderance of maternal sequences. It has recently been shown that fetal cf-DNA sequences are smaller than maternal ones and that the selection of small cf-DNA fragments by size fractionation by agarose gel electrophoresis leads to the enrichment of fetal cf-DNA sequences, thereby permitting the detection of otherwise masked fetal point mutations. In a separate development, the use of MALDI-TOF MS has also been shown to facilitate the detection of fetal point mutations from cf-DNA in maternal plasma. In this study, a combination of these approaches was examined. cf-DNA was extracted from 18 maternal plasma samples, 10 taken at term and 8 obtained early in the second trimester. A total of 41 SNP loci were examined in size-fractionated and total cf-DNA using either a conventional homogeneous MassEXTEND (hME) assay or a nucleotide-specific single allele base extension reaction (SABER) assay. The analysis of total cf-DNA indicated that size fractionation considerably enhanced the sensitivity of the standard hME assay, especially for samples taken early in pregnancy. Size fractionation also rendered the signals obtained by the SABER assay more precise.     

NMR-assisted prediction of RNA secondary structure: identification of a probable pseudoknot in the coding region of an R2 retrotransposon

As the rate of functional RNA sequence discovery escalates, high-throughput techniques for reliable structural determination are becoming crucial for revealing the essential features of these RNAs in a timely fashion. Computational predictions of RNA secondary structure quickly generate reasonable models but suffer from several approximations, including overly simplified models and incomplete knowledge of significant interactions. Similar problems limit the accuracy of predictions for other self-folding polymers, including DNA and peptide nucleic acid (PNA). The work presented here demonstrates that incorporating unassigned data from simple nuclear magnetic resonance (NMR) experiments into a dynamic folding algorithm greatly reduces the potential folding space of a given RNA and therefore increases the confidence and accuracy of modeling. This procedure has been packaged into an NMR-assisted prediction of secondary structure (NAPSS) algorithm that can produce pseudoknotted as well as non-pseudoknotted secondary structures. The method reveals a probable pseudoknot in the part of the coding region of the R2 retrotransposon from Bombyx mori that orchestrates second-strand DNA cleavage during insertion into the genome.     

SNPs Feasibility of Nonlabeled Oligonucletides on Using Electrochemical Sensing

We have demonstrated an electrochemical gene chip protocol for the SNPs detection of nonlabeled DNA. Using an array consisting of streptavidin‐modified gold electrodes, probe DNA were attached through the application of a direct electric field. Electrochemical response changes originating from the hybridization of nucleic acids to protein‐bound nucleic acids using soluble mediators in K₃Fe(CN)₆ solution could then be observed. The electrochemical protocol developed showed high sensitivity and good reproducibility in the detection of DNA hybridization. Significant changes in electrochemical signals were also observed when using target DNA with a single base mismatch, indicating the applicability of this method to single nucleotide polymorphisms (SNPs) detection.     

Simultaneous genotyping of indels and SNPs by mass spectroscopy

Nucleotide insertion/deletion polymorphisms (indels) in ApoE gene were precisely genotyped using artificial ribonucleases and MALDI-TOF MS. The RNA fragments for MS analysis were prepared by treating RNA specimens with our artificial ribonucleases, which consist of LuCl(3) (molecular scissors) and oligonucleotides bearing two acridine groups (RNA-activator for site-selective scission). RNA scission by Lu(III) ion always occurred at the phosphodiester linkages in front of the two acridines, even when the RNA specimens involved consecutive cytidine sequences of different lengths. Thus, even complicated mixtures of these indel specimens were completely genotyped by using only one acridine-bearing oligonucleotide and by subjecting the reaction mixture to single MS measurement. Moreover, single nucleotide polymorphism (SNP) in the consecutive sequences could be genotyped simultaneously with the indels.     

Collisional excitation of vibrations in H2 + H,D encounters using rotational averaging

Vibrational excitation cross sections for H₂ molecular targets colliding with H and D projectiles are computed at several relative energies for the     

1H to 2H uniform cross-polarization nuclear magnetic resonance using 2H Lee-Goldburg irradiation in static powders

Application of conventional cross polarization (CP) to (2)H results in only a narrowband enhancement of the powder line shape due to the quadrupole interaction. We propose a CP scheme to uniformly enhance (2)H spectra in static powders. In this method, a Hartmann-Hahn matched (2)H rf field is applied on the Lee-Goldburg (LG) condition to remove the zeroth-order quadrupole interaction. In order to achieve a uniformly enhanced (2)H powder line shape with a limited (2)H rf intensity, the (1)H rf amplitude in CP is stepwise altered during the contact time. We develop a spin-thermodynamic theory to describe polarization transfer due to CP with LG irradiation (LG-CP) under the influence of the quadrupole interaction, which can successfully reproduce the LG-CP line shapes observed under various experimental conditions. Experimental and simulated (2)H powder spectra are reported for some compounds.