DNA optical sensor: a rapid method for the detection of DNA hybridization

A DNA optical sensor system is proposed based on the combination of sandwich solution hybridization, magnetic bead capture, flow injection and chemiluminescence for rapid detection of DNA hybridization. Bacterial alkaline phosphatase (phoA) gene and Hepatitis B virus (HBV) DNA were used as target DNA. A biotinylated DNA probe was used to capture the target gene onto the streptavidin-coated magnetic beads and a calf intestine alkaline phosphatase (CAP)-labelled DNA probe was used for subsequent enzymatic chemiluminescence detection. The detection cycle was less than 30 min, excluding the DNA hybridization time, which was about 100 min. Both the phoA gene and HBV DNA could be detected at picogramme or femtomole level. No response signal was obtained when target DNA did not exist in the sample. Successive sample detection could be made by removing the magnetic field and a washing step.     

DNA subtraction in situ hybridization--new approaches of detecting genetic defects in surgical pathology specimens

A novel in situ histochemical method of screening genetic alterations of human malignant cells was reported. The method is based on subtraction in situ hybridization of whole genomic DNA from a healthy volunteer or DNA extracted from non-neoplastic tissues. The method can widely examine the possible DNA defects in situ without employing specific probes employing colorimetric reaction in routinely processed surgical pathology materials, i.e. 10% formalin-fixed and paraffin embedded tissue sections. Results can be correlated with histopathological findings. In this report, we also describe the results on genetic alterations of human esophageal squamous cell carcinoma, using this DNA subtraction in situ hybridization.     

Family analysis of human "fingerprints" obtained using a phage M13 DNA probe

The human "fingerprints" detected by the phage M13 DNA probe from 40 simple families with some presumably genetic malformations were observed. When only one parent had a specific band, the mean segregation frequency of all the electrophoretic bands detected was equal to 0.485, which confirmed the hypothesis about the Mendelian inheritance and high level of heterozygosity of hypervariable loci. The mean new measure of similarity between the parents calculated by Li C.C. was 0.54 (this measure within only fathers and only mothers was equal to the mean population level of similarity 0.41).     

Recurrent DNA copy number changes in 1q, 4q, 6q, 9p, 13q, 14q and 22q detected by comparative genomic hybridization in malignant mesothelioma

Comparative genomic hybridization (CGH) analyses were performed on 27 human pleural mesothelioma tumour specimens, consisting of 18 frozen tumours and nine paraffin-embedded tumours, to screen for gains and losses of DNA sequences. Copy number changes were detected in 15 of the 27 specimens with a range from one to eight per specimen. On average, more losses than gains of genetic material were observed. The loss of DNA sequences occurred most commonly in the short arm of chromosome 9 (p21-pter), in 60% of the abnormal specimens. Other losses among the abnormal specimens were frequently detected in the long arms of chromosomes 4 (q31.1-qter, 20%), 6 (q22-q24, 33%), 13 (33%),14 (q24-qter, 33%) and 22 (q13, 20%). A gain in DNA sequences was found in the long arm of chromosome 1 (cen-qter) in 33% of the abnormal specimens. Our analysis is the first genome-wide screening for gains and losses of DNA sequences using comparative genomic hybridization in malignant pleural mesothelioma tumours. The recurrent DNA sequence changes detected in this study suggest that the corresponding chromosomal areas most probably contain genes important for the initiation and progression of mesothelioma.     

Use of DNA fingerprinting for human population genetic studies

DNA fingerprinting techniques have been used in population genetic studies on many different kinds of organisms. Here, we present new applications for multilocus DNA fingerprint probes in population studies and demonstrate the applicability of DNA fingerprinting to human population genetics, using M13 phage DNA as a probe. The new approach, which is based on a factor method of numerical coding of nonquantitative data (factor correspondence analysis-FCA), shows good agreement between population position, as indicated by the three principal factors, and ethnogenetic proximity.     

DNA strand breakage and DNA adducts in lymphocytes of oral cancer patients

In lymphocytes of 12 oral cancer patients (and two control groups) the frequencies of DNA single-strand breaks and DNA-protein cross-linking were determined by alkaline filter elution. We found elevated DNA elution rates, which must be interpreted as an increased strand breakage frequency. There were significant correlations between the DNA strand breakage frequency and smoking habits. Using the 32P-postlabelling assay we determined the DNA adduct level in lymphocytes of 23 oral cancer patients (and two control groups). No significant influence of smoking habit on the DNA adduct level could be detected. There was a significant correlation between the DNA adduct level and the gamma-glutamyltranspeptidase (GGT) value, suggesting systemic influences of alcohol drinking habits on the adduct level.     

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.     

Chemiluminescent microwell hybridization assay for direct detection of human parvovirus B19 DNA

A method for the direct detection of human parvovirus DNA in serum samples that uses a digoxigenin-labeled RNA probe to hybridize with target B19 DNA, followed by capture of the hybrid onto a microtiter plate wells previously coated with a second oligonucleotide probe was developed. The captured hybrid is then detected with anti-digoxigenin-alkaline phosphatase conjugate and chemiluminescent substrate and the reaction read on a scintillation counter. The relative sensitivities of the microwell and standard dot blot hybridization assays were compared. The chemiluminescent microwell hybridization assay was more sensitive than dot-blot hybridization and could be performed in a few hours. This format, therefore, permits rapid and sensitive detection of parvovirus DNA suitable for the clinical setting.     

Endogenous oncornaviral DNA sequences: evidence for two classes of viral DNA sequences in guinea pig cells

The nature of the endogenous viral DNA sequences in guinea pig cells was studied by hybridization. A segment of the viral RNA (r-VRNA) hybridizing to abundant (or reiterated) DNA sequences (R-VDNA) was isolated by recycling to a Cot of 300. The hybridization of the recycled VRNA, as well as the total VRNA, was followed by determining their kinetics and by Wetmur-Davidson analysis. The kinetics of hybridization of total VRNA were complex, did not follow a second-order kinetics, and revealed two slopes by Wetmur-Davidson analysis. The recycled RNA, on the other hand, had a second-order reaction rate expected of the hybridization between a single species of RNA and DNA sequences and yielded a single straight line in a Wetmur-Davidson plot. The Cot1/2 and slope of the recycled r-VRNA was almost identical to that of the abundant VDNA sequences obtained from the hybridization data of the total VRNA. Guinea pig 28S rRNA with or without recycling was used in monitoring hybridization rate. The kinetics of hybridization of 28S RNA followed a second-order reaction and produced a single straight line by Wetmur-Davidson plot, with a second-order reassociation rate constant of 9.6 x 10(-3) liters/mol-s, a Cot1/2 of 104 mol-s/liter, and reiteration frequency of 146. There was no difference in the kinetics of hybridization of 28S RNA before and after recycling. These experiments showed that guinea pig cells contain two classes of VDNA sequences. (i) R-VDNA sequences with a second-order reassociation rate constant of 8.2 x 10(-4) liters/mol-s, a Cot1/2 of 1,219 mol-s/liter, and a reiteration frequency of 12 represent 37.5% of the viral genome. (ii) Unique VDNA sequences with a second-order reassociation rate constant of 1.2 x 10(-4) liters/mol-s, a Cot1/2 of 7,692 mol-s/liter, and a reiteration frequency of 2 represent 62.5% of the viral genome.     

A simple and sensitive DNA hybridization assay used for the routine diagnosis of human parvovirus B19 infection

A dot blot hybridization assay for parvovirus B19 diagnosis was developed by using a PCR-generated probe, digoxigenin labelling, and chemiluminescence detection. Different labelling techniques and hybridization solutions were evaluated. From this analysis a protocol was devised for routine diagnostic use. The protocol enabled 1 pg of B19 DNA to be detected. The results of applying this method to 8,369 diagnostic samples collected during 1994 and 1995 are given.     

Homologous pairing in stretched supercoiled DNA

By using elastic measurements on single DNA molecules, we show that stretching a negatively supercoiled DNA activates homologous pairing in physiological conditions. These experiments indicate that a stretched unwound DNA locally denatures to alleviate the force-driven increase in torsional stress. This is detected by hybridization with 1 kb of homologous single-stranded DNA probes. The stretching force involved (approximately 2 pN) is small compared with those typically developed by molecular motors, suggesting that this process may be relevant to DNA processing in vivo. We used this technique to monitor the progressive denaturation of DNA as it is unwound and found that distinct, stable denaturation bubbles formed, beginning in A+T-rich regions.     

Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.     

DNA typing of human leukocyte antigen sequence polymorphisms by peptide nucleic acid probes and MALDI-TOF mass spectrometry

A novel analytical method using PNA probes detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied to type sequence polymorphisms within the human leukocyte antigen (HLA), DQA locus. Streptavidin-coated magnetic beads were used to immobilize biotinylated DNA. PNA probes representing possible alleles were then prepared for the immobilized DNA hybridization. The nonspecific PNA probes were removed with stringent washes. The PNA/DNA/beads conjugate was analyzed by MALDI-TOFMS. The genotype of the DNA was determined by the detected molecular masses of the released PNA probes. Reproducible and accurate genotyping was achieved by this analytical method.     

Frequency of the delta ccr5 deletion allele in the urban Brazilian population

Studies on screening genes conferring resistance to HIV-1 and AIDS onset have shown a direct relationship between a 32 base pair (bp) deletion in the CCR5 beta-chemokine receptor gene (delta ccr5 mutant allele) and long survival of HIV-1 infected individuals bearing this mutation. These findings led to an interest in studies of delta ccr5 allele distribution in human populations. In the present study, polymerase chain reactions (PCR) in genomic DNA samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 193-bp product from the normal CCR5 allele and a 161-bp product from the 32-bp deletion allele. In an investigation of the urban Brazilian population we detected a 93% frequency of normal CCR5/CCR5 homozygous individuals and a 7% frequency of CCR5/delta ccr5 heterozygous individuals. The frequency of the delta ccr5 mutant allele in this population is 0.035; however, no homozygous delta ccr5 individual has been detected thus far. This is the first evidence for the contribution of the delta ccr5 allele to the genetic background of the urban Brazilian population, which is characterized by intense ethnic admixture. These findings open perspectives for further studies on the relationship between delta ccr5 allele frequency and AIDS onset in high-risk HIV-1 exposures individuals.     

Naturally occurring variation in bristle number and DNA polymorphisms at the scabrous locus of Drosophila melanogaster

The association between quantitative genetic variation in bristle number and molecular variation at a candidate neurogenic locus, scabrous, was examined in Drosophila melanogaster. Approximately 32 percent of the genetic variation in abdominal bristle number (21 percent for sternopleural bristle number) among 47 second chromosomes from a natural population was correlated with DNA sequence polymorphisms at this locus. Several polymorphic sites associated with large phenotypic effects occurred at intermediate frequency. Quantitative genetic variation in natural populations caused by alleles that have large effects at a few loci and that segregate at intermediate frequencies conflicts with the classical infinitesimal model of the genetic basis of quantitative variation.     

Diagnosis of avian adenovirus infections using DNA in situ hybridization

Three DNA oligonucleotide probes designated FN-23, FN-48, and FN-96 were evaluated for the diagnosis of aviadenovirus infections by DNA in situ hybridization. Paraffin-embedded tissues were obtained from birds with confirmed adenovirus infection, birds with putative adenovirus infections, and birds with intranuclear inclusions caused by herpesvirus and polyomavirus. In birds with confirmed adenovirus infection, probes FN-23 and FN-96 identified 78% and 72% of diseased individuals, respectively. Only probe FN-48 detected chickens with group II adenovirus infection. In birds with putative adenovirus infection, the DNA probes confirmed aviadenovirus infections in 76% of the population. Probes FN-23, FN-96, and FN-48 detected 85%, 74%, and 18% of adenovirus-infected birds, respectively. None of the DNA probes cross-hybridized with tissues from polyomavirus-infected psittaciform birds or with tissues from a chicken with infectious laryngotracheitis. In contrast, probe FN-23 did cross-hybridize to herpesvirus-infected tissues from two of eight psittaciform birds with Pacheco's parrot disease. Probes FN-48 and FN-96 did not react with these tissues.     

Extent of heterogeneity in mitochondrial DNA of European populations

Variation in the mitochondrial DNA (mtDNA) control region as detected by sequence-specific oligonucleotide (SSO) probes is described for 595 individuals from six European or European-derived populations. Estimates of diversity for mtDNA types exceed 0.91 in all populations, while 50% of the 158 types which were observed occur only once. Of 68 shared types, most occur rarely (< 3% of the total population); only one type occurs at a frequency greater than 10%, and it is present at comparable frequencies in all six populations (18-29%). An analysis of molecular variance (AMOVA) incorporating genetic distances between types shows that 100% of the variation present in the total sample is attributable to within-population diversity, while there are essentially no between-population differences. Another AMOVA was performed for the first hypervariable region SSO sites only, which included this sample plus an additional 537 SSO types from mine more European populations that were inferred from published mtDNA control region sequence data. Similar results were obtained, with over 99% of the variation overall attributable to within-population differences, and less than 1% of the variation attributable to between-population differences. The Saami were the most different from other populations, which had been observed in an earlier study of nucleotide sequence data. Overall, there is no statistically significant heterogeneity for European populations (p > 0.001), and these groups are virtually indistinguishable with respect to mtDNA SSO types. These results demonstrate the utility of mtDNA typing for forensic investigations.     

DNA fingerprinting for genetic monitoring of inbred laboratory rats and mice

DNA fingerprinting using a nonisotopically labeled minisatellite probe provided a valuable technique for genetic monitoring/quality control of laboratory rodents. Each of 12 inbred rat strains had a unique fingerprint pattern, and colonies separated for over 20 years had identical or nearly identical patterns. Strain LOU/Iap, which is known to have been genetically contaminated in the past, was clearly different from strain LOU/CN, supporting previous findings of studies using biochemical markers. Inbred strains of mice were also found to differ from each other. The F1 hybrid between C57BL/6 and CBA/Ca could not be distinguished from C57BL/6 by using DNA fingerprints, although they could be distinguished by using biochemical markers. Some congenic strains differed from their inbred partner. A suspected genetic contamination of MRL/Mp-lpr mice could not be detected in a sample of the breeding colony by using biochemical markers; however, DNA fingerprints from the suspect animals clearly demonstrated genetic segregation. DNA fingerprinting will be of particular value in investigating suspected problems as only a small sample of fresh, frozen, or ethanol-preserved tissue is needed. Thus, the actual suspect animals can be studied, rather than samples from a breeding colony from which contaminated animals may already have been eliminated.     

Sequence-selective biosensor for DNA based on electroactive hybridization indicators

Deoxyribonucleic acid was covalently immobilized onto oxidized glassy carbon electrode surfaces that had been activated using 1-[3-(dimethylamino)-propyl]-3-ethylcarbodimide hydrochloride and N-hydroxysulfosuccinimide. This reaction is selective for immobilization through deoxyguanosine (dG) residues. Immobilized DNA was detected voltammetrically, using tris (2,2'-bipyridyl)cobalt(III) perchlorate and tris (1,10-phenanthroline)cobalt(III) perchlorate (Co(bpy)3(3+) and Co(phen)3(3+). These complexes are reversibly electroactive (1e-) and preconcentrate at the electrode surface through association with double-stranded DNA. Voltammetric peak currents obtained with a poly(dG)poly(dC)-modified electrode depend on [Co(bpy)3(3+)] and [Co(phen)3(3+)] in a nonlinear fashion and indicate saturation binding with immobilized DNA. Voltammetric peak currents for Co(phen)3(3+) reduction were used to estimate the (constant) local DNA concentration at the modified electrode surface; a binding site size of 5 base pairs and an association constant of 1.74 x 10(3) M(-1) yield 8.6 +/- 0.2 mM base pairs. Cyclic voltammetric peak separations indicate that heterogeneous electron transfer is slower at DNA-modified electrodes than at unmodified glassy carbon electrodes. A prototype sequence-selective DNA sensor was constructed by immobilizing a 20-mer oligo (deoxythymidylic acid) (oligo(dT)20), following its enzymatic elongation with dG residues, which yielded the species oligo(dT)20(dG)98. Cyclic voltammograms of 0.12 mM Co(bpy)3(3+) obtained before and after hybridization with poly-(dA) and oligo(dA)20 show increased cathodic peaks after hybridization. The single-stranded form is regenerated on the electrode surface by rinsing with hot deionized water. These results demonstrate the use of electroactive hybridization indicators in a reusable sequence-selective biosensor for DNA.