Discrimination between Atlantic and Pacific Subspecies of Northern Bluefin Tuna (Thunnus thynnus) by Magnetic-Capture Hybridization Using Bacterial Magnetic Particles

The previously developed magnetic-capture hybridization technique employing bacterial magnetic particles was applied to discriminate between Atlantic and Pacific subspecies of the northern bluefin tuna (Thunnus thynnus) using specific DNA sequences. Nucleotide sequences of a 925-bp fragment (ATCO) flanking the mitochondrial ATPase and cytochrome oxidase subunit III genes in these two subspecies were compared. Two regions having single-nucleotide and three-nucleotide differences between the subspecies were adopted to design DNA probes (NR1, 21-mer; NR2, 29-mer), and two internal primer sets were designed to amplify DNA fragments containing these regions. The DNA probes were immobilized on bacterial magnetic particles via streptavidin-biotin conjugation and subjected to magnetic-capture hybridization with the digoxigenin-labeled fragments amplified using the internal primers. The luminescence intensities of DNA on bacterial magnetic particles obtained by hybridization between the probes and the complementary fragments were higher than those obtained by hybridization with noncomplementary fragments. These data suggest that this system employing DNA on bacterial magnetic particles may be useful for discrimination of these two subspecies by recognizing a single-nucleotide difference. Received January 17, 2000; accepted January 28, 2000.     

Germline mutations of the RET proto-oncogene in eight Japanese patients with multiple endocrine neoplasia type 2A (MEN2A)

Multiple endocrine neoplasia type 2A (MEN2A) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. The gene responsible for MEN2A was localized by linkage analysis to chromosome 10q11.2 in 1987, and recently mutations in RET, a proto-oncogene in the candidate region, were discovered in patients with MEN. The majority of mutations found so far in MEN2A patients have been located in nucleotide sequences encoding cysteine residues in the extracellular domain of RET. To characterize MEN2A germline alterations in the Japanese population, we screened DNA from eight unrelated patients for mutations in exons 10 and 11 of the RET proto-oncogene and found mutations in all eight patients, at codons 618, 620, or 634; each of these sites encodes a cysteine residue in the extracellular domain of RET. The mutations were confirmed in other affected individuals in the respective families by digestion of polymerase chain reaction (PCR) products containing the mutated codons with restriction enzymes (RsaI, CfoI, or AluI) for which cleavage sites had been generated by the specific genetic alteration. These PCR-restriction enzyme systems will be useful for genetic diagnosis in members of families carrying these mutations.     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

High-Density Nucleoside Analog Probe Arrays for Enhanced Hybridization

Abstract

DNA probe arrays were synthesized with analogs of 2,6-diaminopurine and 2′-O-methyl-thymidine in place of A and T. AT-rich GeneChip® test arrays containing 14-mer or 20-mer analog probes improved hybridization to fluorescently-labeled RNA sequences under stringent conditions.     

Detection of known mutations in hypertrophic cardiomyopathy using oligonucleotide microarrays assisted by improved base stacking hybridization

With the assistance of improved base stacking hybridization, a low-density microarray, containing 12 capture probes, was used to identify 7 known hypertrophic cardiomyopathy-related mutations in the gene of MYH7 (-myosin heavy chain). The hybridization targets, amplified from 11 plasmids containing wild type or mutation sequences of MYH7 and healthy genomic DNA, were prepared by single-step fluorescence labeled asymmetric PCR. Six single base substitutions and a trinucleotide deletion were identified unambiguously, and the average discrimination ratio (Q_mut) for artificial heterozygous samples was as high as 16.2.     

Development of a PCR/Ligase Detection Reaction/Nanogold-Based Universal Array Approach for the Detection of Low-Abundant DNA Point Mutations

The aim of this study was to investigate the feasibility of combining PCR and ligase detection reaction (LDR) with a novel nano-gold-based universal array for the detection of low abundance point mutations from fetal DNA in maternal plasma samples. The sequence with the target point mutation was first amplified by PCR and then used as a template for LDR in which the upstream specific primer contains a tag sequence at the 5′-end. After hybridization to the probes of a universal array containing anti-tag sequences, the ligated products were bound to streptavidin-labeled nano-gold particles and the hybridization signals were amplified by silver staining. The PCR/LDR/universal array was first tested for sensitivity with nano-gold-based detection, and then this system was applied to detect the low abundance specific mutation IVS2 654(C→T) of the β-globin gene in a model using maternal plasma samples. The nano-gold-based method unambiguously identified a single mutation at a sensitivity of 1:1000. This approach was applied to detect the paternally inherited IVS2 654(C→T) mutation from thirty maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. The PCR/LDR/nano-gold-based universal array is able to detect low-abundance point mutations with high sensitivity.     

The synergy of germline C634Y and V292M RET mutations in a northern Chinese family with multiple endocrine neoplasia type 2A

Genetic analysis for germline mutations of RET proto‐oncogene has provided a basis for individual management of medullary thyroid carcinoma (MTC) and pheochromocytoma. Most of compound mutations have more aggressive phenotypes than single point mutations, but the compound C634Y/V292M variant in MTC has never been reported. Thus, we retrospectively investigated synergistic effect of C634Y and V292M RET germline mutations in family members with multiple endocrine neoplasia type 2A. Nine of 14 family members in a northern Chinese family underwent RET mutation screening using next‐generation sequencing and PCR followed by direct bidirectional DNA sequencing. Clinical features of nine individuals were retrospectively carefully reviewed. In vitro, the scratch‐wound assay was used to investigate the difference between the cells carrying different mutations. We find no patients died of MTC. All 3 carriers of the V292M variant were asymptomatic and did not have biochemical or structural evidence of disease (age: 82, 62 and 58). Among 4 C634Y mutation carriers, 2 patients had elevated calcitonin with the highest (156 pg/mL) in an 87‐year‐old male. Two carriers of compound C634Y/V292M trans variant had bilateral MTC with pheochromocytoma or lymph node metastasis (age: 54 and 41 years, respectively). Further, the compound C634Y/V292M variant had a faster migration rate than either single point mutation in vitro (P < .05). In conclusion, the V292M RET variant could be classified as ‘likely benign’ according to ACMG (2015). The compound variant V292M/C634Y was associated with both more aggressive clinical phenotype and faster cell growth in vitro than was either single mutation.     

Microgravimetric DNA sensor based on quartz crystal microbalance: comparison of oligonucleotide immobilization methods and the application in genetic diagnosis

We report on the study of immobilization DNA probes onto quartz crystal oscillators by self-assembly technique to form variety types of mono- and multi-layered sensing films towards the realization of DNA diagnostic devices. A 18-mer DNA probe complementary to the site of genetic beta-thalassaemia mutations was immobilized on the electrodes of QCM by covalent bonding or electrostatic adsorption on polyelectrolyte films to form mono- or multi-layered sensing films by self-assembled process. Hybridization was induced by exposure of the QCMs immobilized with DNA probe to a test solution containing the target nucleic acid sequences. The kinetics of DNA probe immobilization and hybridization with the fabricated DNA sensors were studied via in-situ frequency changes. The characteristics of QCM sensors containing mono- or multi-layered DNA probe constructed by direct chemical bonding, avidin-biotin interaction or electrostatic adsorption on polyelectrolyte films were compared. Results indicated that the DNA sensing films fabricated by immobilization of biotinylated DNA probe to avidin provide fast sensor response and high hybridization efficiencies. The effects of ionic strength of the buffer solution and the concentration of target nucleic acid used in hybridization were also studied. The fabricated DNA biosensor was used to detect a set of real samples. We conclude that the microgravimetric DNA sensor with its direct detection of amplified products provide a rapid, low cost and convenient diagnostic method for genetic disease.     

Cloning and sequencing of the Clostridium perfringens enterotoxin gene

Several gene banks of Clostridium perfringens in E. coli were constructed. Using a mixture of synthetic 29-mer DNA probes clones were selected containing inserts from the C. perfringens gene coding for the enterotoxin. This has allowed sequencing of the complete gene and its flanking regions. The decuded amino acid sequence (320 a.a.) was found to differ at several sites from the sequence published previously by others. Two 40-mer DNA-probes were used to detect the toxin gene in C. perfringens strains isolated from the faeces of different non-symptomatic animals. Only 6% of the strains were found to possess the gene.     

Rapid F508del and F508C assay using fluorescent hybridization probes

Amplification and fluorescent genotyping of the cystic fibrosis F508del locus was achieved from human genomic DNA in less than 30 min. The hybridization of adjacent fluorescent probes at the mutation site was monitored by resonance energy transfer between fluorescein and Cy5 during heating or cooling. Characteristic curves were obtained for each genotype; the first derivative of these fluorescent curves has a maximum at an apparent hybridization temperature (Tm) that is specific for each probe/allele duplex. The direction and rate of temperature change determines the difference between the apparent Tm and the true equilibrium Tm. One hundred and five sample were genotyped for the F508del cystic fibrosis mutation by heating and cooling curve profiles. These genotypes were validated by allele-specific amplification. Two fluorescein hybridization probes were designed to match the wild-type sequence perfectly from either codons 502 to 513 or from 504 to 511 on the cystic fibrosis transconductance regulator gene of chromosome 7. While genotyping for the F508del, an allele with the F508C base change was detected. For both F508del and F508C variants, the Tm shift from wild type was greater with a 24-mer probe than with a 35-mer probe. Fluorescent monitoring of hybridization probes is a versatile technique that can detect unexpected sequence alterations.     

Comparative modeling and analysis of microfluidic and conventional DNA microarrays

A theoretical analysis was developed to predict molecular hybridization rates for microarrays where samples flow through microfluidic channels and for conventional microarrays where samples remain stationary during hybridization. The theory was validated by using a multiplexed microfluidic microarray where eight samples were hybridized simultaneously against eight probes using 60-mer DNA strands. Mass transfer coefficients ranged over three orders of magnitude where either kinetic reaction rates or molecular diffusion rates controlled overall hybridization rates. Probes were printed using microfluidic channels and also conventional spotting techniques. Consistent with the theoretical model, the microfluidic microarray demonstrated the ability to print DNA probes in less than 1 min and to detect 10-pM target concentrations with hybridization times in less than 5 min.     

Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface

Here, we describe a multi-parametric study of DNA hybridization to probes with 20–70% G + C content. Probes were designed towards 71 different sites/mutations in the phenylalanine hydroxylase gene. Seven probe lengths, three spacer lengths and six stringencies were systematically varied. The three spacer lengths were obtained by placing the gene-specific sequence in discrete steps along the 60-mer probes. The study was performed using Agilent 8 × 15 000 probes custom-made arrays and a home-built array washer providing different stringencies to each of the eight sub-arrays on the slides. Investigation of hybridization signals, specificity and dissociation curves indicated that probes close to the surface were influenced by an additional stringency provided by the microarray surface. Consistent with this, probes close to the surface required 4 × SSC, while probes placed away from the surface required 0.35 × SSC wash buffers in order to give accurate genotyping results. Multiple step dissociation was frequently observed for probes placed furthest away from surface, but not for probes placed proximal to the surface, which is consistent with the hypothesis that there is different stringency along the 60-mer. The results have impact on design of probes for genotyping, gene expression and comparative genome hybridization analysis.     

The sensitivity of activated Cys Ret mutants to glial cell line-derived neurotrophic factor is mandatory to rescue neuroectodermic cells from apoptosis

Hirschsprung's disease (HSCR), a frequent developmental defect of the enteric nervous system is due to loss-of-function mutations of RET, a receptor tyrosine kinase essential for the mediation of glial cell-derived neurotrophic factor (GDNF)-induced cell survival. Instead, gain-of-function Cys mutations (e.g., Cys(609), Cys(620), and Cys(634)) of the same gene are responsible for thyroid carcinoma (MEN2A/familial medullary thyroid carcinoma) by causing a covalent Ret dimerization, leading to ligand-independent activation of its tyrosine kinase. In this context, the association of Cys(609)- or Cys(620)-activating mutations with HSCR is still an unresolved paradox. To address this issue, we have compared these two mutants with the Cys(634) Ret variant, which has never been associated with HSCR, for their ability to rescue neuroectodermic cells (SK-N-MC cells) from apoptosis. We show here that despite their constitutively activated kinase, the mere expression of these three mutants does not allow cell rescue. Instead, we demonstrate that like the wild-type Ret, the Cys(634) Ret variant can trigger antiapoptotic pathways only in response to GDNF. In contrast, Cys(609) or Cys(620) mutations, which impair the terminal Ret glycosylation required for its insertion at the plasma membrane, abrogate GDNF-induced cell rescue. Taken together, these data support the idea that sensitivity to GDNF is the mandatory condition, even for constitutively activated Ret mutants, to rescue neuroectodermic cells from apoptosis. These findings may help clarify how a gain-of-function mutation can be associated with a developmental defect.     

Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin     

Immobilization and hybridization of DNA in a sugar polyacrylate hydrogel

Using a non-contact microarrayer, amine-terminated probe oligonucleotides representing 20-, 50-, and 70-mer fragments of the fliC gene were covalently coupled into three-dimensional regions in a "sugar polyacrylate" hydrogel based on poly(6-acryloyl-beta-O-methyl galactopyranoside-co-aminopropyl methacrylamide). The arrayer deposited the solution containing ssDNA probes in discrete regions on the surface of the gel (i.e. as a droplet with a ca. 450 microm diameter), allowing penetration and attachment of the ss DNA within the three dimensional region of the gel. The attachment was mediated by the homobifunctional crosslinker bis-succinimidyl suberate. Confocal microscopy showed the density of attached probe DNA was greatest in the interior-most regions of the gel volume. Target ssDNA (20- and 70-mer) was able to diffuse through the gel and undergo successful hybridization with the probes. For target ssDNA in the concentration range 0.19 microM to 6.0 microM, there was a linear correlation between DNA concentration and the fluorescence of the gel region where hybridization occurred.     

Detection of a single base substitution in a single cell using the LightCycler

For known mutations, real time polymerase chain reaction followed by melting curve analysis, using hybridization probes, is highly sensitive, rapid and an efficient approach to mutation detection. We have used this approach on the LightCycler for the detection of single base mutations in a single cell, without nested PCR. Hybridization probes were designed for two sequences in the BRCA1 gene containing a single base substitution and deletion, respectively. Polymerase chain reactions of small fragments (100-200 bp) containing the probe sequences were optimized using SYBR Green1, before using hybridization probes. The 5'-probes were 3'-labeled with FITC, whereas the 3'-probes, covering the mutation, were 5'-labeled with LC-Red640 (wild type probes) or LC-Red705 (mutant probes). Dual color detection of wild type and mutant sequences in a single tube was tested on single cells. The reaction mix was prepared in reaction capillaries and a single cell, picked by micromanipulation, was added to this mix. The DNA from the cell is released during the 5-min preheating step of the PCR, using the FastStart hybridization kit (Roche). Reproducible results were obtained, without the need of nested PCR. The technique is useful for microdissected tumors and, with other genes, has great potential for pre-implantation diagnosis in IVF and analysis of residual disease in cancer.     

Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB

Background and Aims: Fluoroquinolone‐containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87and 91 of GyrA among fluoroquinolone‐resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Materials & Methods: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Results: Norfloxacin‐resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin‐resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Conclusion: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.     

Classification of rice germplasm. I. Analysis using ALP and PCR-based RFLP

The potential of using a PCR-based approach to detect DNA polymorphism for rice germplasm classification was compared with that of Southern-based RFLP analysis. Thirty-five Iranian rice varieties were studied along with 2 typical Indica and 3 typical Japonica varieties. Thirteen mapped RFLP markers were used as hybridization probes against Southern blots containing digests of one restriction endonuclease; 12 of the 13 probes detected polymorphism in the varieties. Fifteen sets of oligonucleotides derived from sequences near the ends of the same probes and of two other mapped probes were used as primers for PCR amplification of total genomic DNA of the varieties. Amplicon length polymorphisms (ALPs) were detected with 6 of the 15 sets of primers. To identify additional polymorphism, the PCR products were digested with nine different restriction endonucleases recognizing 4- or 5-bp DNA sequences and analyzed by gel electrophoresis in agarose and polyacrylamide. RFLPs were detected for 11 sets of primers, due to point mutations and to addition/deletion events that were too small to be detected as ALPs. Because PCR products are easily generated and may be analyzed in detail through the use of restriction endonucleases that cut rice DNA frequently, PCR-based RFLP analysis is a useful tool for the classification of rice germplasm.     

Amplification of repeated DNA sequences in wheat X rye hybrids regenerated from tissue culture

Summary Previous C-banding analysis of wheat (Triticum aestivum)X rye (Secale cereale) hybrids regenerated from tissue culture revealed enlarged C-bands in some rye chromosomes, but the molecular nature of the change was not determined. In situ hybridization using two DNA probes containing repeated sequences from rye telomeric heterochromatin was conducted on these wheatX rye hybrids and their progeny to investigate the occurrence of amplification in repeated sequences. Clones pSC 74 and pSC 119, which contain sequences from the 480-bp and 120-bp repeated DNA families of rye, respectively, were used as probes. Amplification of 480-bp repeated sequences in the short arm telomere of chromosome 7R was detected in three wheatxrye hybrids and their progeny. The amplified 480-bp sequences were detected by an enlarged hybridization site for pSC 74 at the 7RS telomere, and by the appearance at this same telomeric site of an unlabeled, blue chromosome segment in an otherwise completely brown chromosome hybridizing entirely to the biotin-labeled pSC 119 probe. This variant form of chromosome 7R was not observed in several Chaupon plants, or in the other hybrids derived from the same embryos, indicating the origin of the change in tissue culture. The amplified sequences were inherited up to at least three generations. Deletions and translocations were also observed.Contribution No. 87-9-J, Kansas Agricultural Experiment Station, Kansas State University     

Genomic mutation rates: what high‐throughput methods can tell us

High‐throughput DNA analyses are increasingly being used to detect rare mutations in moderately sized genomes. These methods have yielded genome mutation rates that are markedly higher than those obtained using pre‐genomic strategies. Recent work in a variety of organisms has shown that mutation rate is strongly affected by sequence context and genome position. These observations suggest that high‐throughput DNA analyses will ultimately allow researchers to identify trans‐acting factors and cis sequences that underlie mutation rate variation. Such work should provide insights on how mutation rate variability can impact genome organization and disease progression.