DNA probe hybridisation in microwells using a new bioluminescent system for the detection of PCR-amplified HIV-1 proviral DNA

A new bioluminescent detection system combined with a sandwich DNA hybridisation reaction in microwells has been developed for the detection of human immunodeficiency virus type 1 (HIV-1) provirus DNA amplified by the polymerase chain reaction (PCR). First, a fragment of the HIV-1 gag gene was amplified. The amplified DNA fragments were denatured and hybridised to a capture probe immobilised in microwells and to a biotinylated detection probe. A streptavidin-pyruvate kinase conjugate could then react on the biotinylated probe and the kinase activity detected by means of the luciferin-luciferase system, with production of a bioluminescent signal. This sandwich assay followed by a bioluminescent reaction detected as little as 7 amol of target DNA. The bioluminescent assay detected 5 HIV copies generated after one round of PCR, even if no band was seen on an agarose gel. The assay was applied to the detection of HIV-proviral DNA in peripheral blood mononuclear cells after one round of PCR and allowed to clearly identify a positive sample as compared to nested PCR.     

In situ hybridization detection of short viral amplicon sequences within cultured cells and body fluids after the in situ polymerase chain reaction

Using single primer pairs, intracellular gene sequences of cytomegalovirus (CMV-Towne's strain) and alpha-tubulin were amplified (in situ PCR) from cells in human body fluids and in suspensions. Visualization of CMV amplificants was carried out by in situ hybridization (ISH), using both a biotinylated double-stranded DNA probe and a radiolabelled oligonucleotide probe. Visualization of alpha-tubulin amplificants was achieved using both radiolabelled single-stranded cRNA and oligonucleotide probes. Liberated amplificants were also identified by bands of expected size by gel electrophoresis. The specificity of the PCR products was confirmed by Southern blot analysis. Intracellular amplification was identified both in unfixed cells and, optimally, after brief alcohol fixation, whilst maintaining relative isotonicity in all working solutions. For CMV, enhanced signal was observed in cells (cultured fibroblasts or urine sediment) undergoing in situ PCR using either biotinylated or radiolabelled probes compared with controls undergoing ISH alone. For alpha-tubulin, radiolabelled riboprobes and oligoprobes only produced signals within cells (human peripheral lymphocytes, ascitic fluid and bladder washings from routine cytological specimens) after in situ PCR, but not after ISH alone. Morphological evaluation was superior with biotinylated probes, and minimal back-diffusion effect was found compared with radiolabelled probes. Up to 80% of cells survived thermal cycling. In situ PCR detected short sequence (100 bp) foreign DNA and low copy number genomic DNA, and was superior to ISH alone. In contrast to radiolabelled probes, very small CMV amplificants could be detected without a significant 'back-diffusion' effect when using the large biotinylated probe in this model system.     

A method to increase the sensitivity of mutation specific oligonucleotide hybridization using asymmetric polymerase-chain reaction (PCR)

We propose a simple and reliable method to increase the sensitivity of mutation specific oligonucleotide hybridization (MSOH) at least 2.5 times, when it is used to detect mutations in samples of DNA from tumor tissues. The method is based on using single stranded (ss) DNA, amplified by asymmetric PCR, as a target for MSOH analysis. During the first step, genomic DNA, isolated from tissue samples, has to be amplified by "standard", symmetric PCR, with sense and antisense primers in equimolar concentration. This amplification can be performed in a diminished volume of reaction mixture. In the second step obtained double stranded (ds) PCR DNA-product can be used as a template for asymmetric PCR, using only a single primer. The ss DNA must be complementary to the set of mutation specific oligonucleotides. By this innovation we have been able to clarify questionable results of MSOH using ds DNA as a target. Comparing MSOH from ss DNA to that from ds DNA, the observed rate of Gs-alpha mutations in thyroid tumor tissue samples increased to 16.7% (14/66) from 6% (4/66).     

Lack of point mutation of the APP gene in sporadic Alzheimer's disease in Japanese

We investigated point mutations of the APP gene in 66 patients with sporadic Alzheimer's disease (AD) and 180 normal individuals by use of the PCR (polymerase chain reaction) method. Both the AD patients and the normal individuals were Japanese. We extracted DNA from blood samples using the phenol-chloroform method and amplified exons 16 and 17 of the APP gene by PCR. PCR products were digested by MBO-II (exon 16) and BCL-1(exon 17). Electrophoresis was carried out with 3% agarose gel and the separated fragments were stained with ethidium bromide. In addition we investigated other point mutations of exons 16 and 17 by use of the PCR-SSCP (single stranded conformation polymorphisms) method, and found no fragments that exhibited point mutations in the AD patients and normal individuals. These findings indicate that the presence of point mutation of the APP gene is not a major cause of AD in the Japanese population.     

Isolation of murine telomere-proximal sequences by affinity capture and PCR

We describe a method of selectively enriching for murine telomere-proximal sequences using affinity capture followed by PCR amplification. The telomeric fragments were selected from NotI-digested and lambda exonuclease-resected mouse genomic DNA by annealing to a biotinylated riboprobe containing multiple copies of the telomere repeat (TTAGGG)n. The resultant DNA-RNA hybrids were selectively retained on a matrix with covalently bound avidin. The captured DNA was then specifically released by ribonuclease action, and PCR amplification was performed using mouse repeat primers. The PCR products were cloned and used to screen a mouse genomic cosmid library, and the resultant cosmid clones were analyzed by fluorescence in situ hybridization. Ten of 70 clones analyzed gave telomere-proximal hybridization signals, indicating an at least 500-fold enrichment for telomere-proximal sequences.     

Alterations in cell cycle regulation in mouse skin tumors

Using a modified tip of the atomic force microscope (AFM), we harvested several strands of genomic DNA from a nanometer region of mouse chromosomes. We have also co-developed a random PCR method to amplify the recovered genomic DNA, in which a single DNA molecule of several kilobasepairs could be amplified efficiently. A subsequent fluorescence in situ hybridization (FISH) indicated that the amplified DNA originally came from the tip-manipulated regions of mouse chromosomes. Several fragments containing unique sequences were identified using Southern hybridization after subcloning the PCR products into pUC18 plasmid. The present results showed a potential application of AFM to genomic analysis.     

A biotinylated MutS fusion protein and its use in a rapid mutation screening technique

The Escherichia coli DNA mismatch repair protein, MutS, binds single base pair mismatches and short deletions in vivo and in vitro. To adapt this protein for mutation detection, a fusion protein of E. coli MutS with a biotinylated peptide domain has been constructed (MutSb). The biotinylation tag facilitates MutS detection and binding by avidin without significantly altering the DNA mismatch binding properties of MutS in vitro. We describe a novel and rapid mutation detection method with MutSb using streptavidin-coated magnetic beads and demonstrate that MutSb can also be used to remove mismatch containing DNA fragments from a mixture of DNA fragments in solution.     

Mucoepidermoid carcinoma arising in Warthin''s tumour of the parotid gland: report of two cases with histopathological, ultrastructural and immunohistochemical studies

OBJECTIVE: To study the molecular defects of congenital adrenal hyperplasia (CAH). STUDY DESIGN: Twenty Chinese patients, including 8 with salt-wasting (SW) type CAH, 11 with simple virilizing (SV) type CAH and 1 with nonclassical (NC) type CAH, were recruited. Two rounds of the polymerase chain reaction (PCR) were used to study the 21-hydroxylase gene (CYP21). The primary PCR amplified CYP21-specific DNA fragments, and the secondary PCR used products from the primary PCR for analysis of amplification-created restriction sites (ACRS) and direct DNA sequencing. In all patients, ACRS analysis was done at 12 possible mutation sites, and then direct DNA sequencing was performed to confirm or define the molecular defects. RESULTS: Ten different mutations, including nine point mutations and gross gene deletion or conversion, were found in this study. Of the nine point mutations, eight could be easily detected by ACRS analysis. The three most common mutations were codon (CD)172 t-->a (I172N), IVS-II 656 c/a-->g, and gross gene deletion or conversion, accounting for 27.5% (11/40 alleles), 25% (10/40) and 20% (8/40) of all identified mutations, respectively. All SW patients were compound heterozygotes of IVS-II 656, gross gene deletion or conversion, or other severe defects, including CDs236 (t-->a) (I236N)+ 237 (t-->a) (V237E)+ 239 (t-->a) (M239K), CD306 (+t), CD318 (c-->t) (Q318X) and CD356 (c-->t) (R356W) mutations. All SV patients had one allele with a CD172 (I172N) mutation. One allele of an NC patient had a CD183 (c-->g) (D183E) mutation, and the other allele was not defined. In the whole series, four alleles (10%) had more than one mutation. CONCLUSION: We found 10 different mutations in this study. The correlation between genotypes and phenotypes was compatible with the reported data. Two rounds of PCR and ACRS analysis may provide important information for genetic counseling, prenatal diagnosis and management of families at risk for CAH.     

Novel DNA detection system of flow injection analysis (2). The distinctive properties of a novel system employing PNA (peptide nucleic acid) as a probe for specific DNA detection

In order to realize immediate detection of a double stranded DNA amplified by Polymerase Chain Reaction (PCR), we applied Peptide Nucleic Acid (PNA) to the probe of DNA detection system using Surface Plasmon Resonance (SPR). We report our success in immediate detection of PCR products solution with high sequence-specificity.     

A method for high yield isolation and purification of anti-native DNA antibodies present in lupus sera

Antibodies to nucleic acids may serve as biochemical tools or as probes of cellular function. Particularly important, but also particularly difficult to obtain, is antibody which reacts exclusively with double stranded DNA. We describe here a method for the separation of antibodies to double stranded DNA from SLE serum, using hydroxyapatite to which DNA is adsorbed at a low molarity of phosphate buffer. Having applied the serum to the column we passed it through a continuous gradient of phosphate buffer ranging from 0.005 to 0.5 M. Deoxyribonuclease and magnesium ions were added when the gradient had reached the molarity at which single stranded DNA had already been desorbed and double stranded DNA began to be eluted. The antibody to native DNA that we obtained reacted in complement fixation, counterimmunoelectrophoresis and Farr's assay with native DNA and did not react with single stranded DNA, single and double stranded RNA or with a panel of 24 protein-coupled nucleosides, nucleotides and dinucleotides.     

Sanger DNA-sequencing reactions performed in a solid-phase nanoreactor directly coupled to capillary gel electrophoresis

A miniaturized, solid-phase nanoreactor was developed to prepare Sanger DNA-sequencing ladders which was directly interfaced to a capillary gel electrophoresis system. A biotinylated fragment of the rat brain actin gene (1 kbp) was amplified by PCR and attached to the interior wall of an (aminoalkyl)silane-derivatized fused-silica capillary tube via a biotin/streptavidin/biotin linkage. Coverage of the capillary wall with the biotinylated DNA averaged 77 +/- 10%. Stability of the anchored template under pressure (33 nL/s) and electroosmotic flows (11.3 nL/s) were favorable, requiring rinsing for > 150 h to reduce the surface coverage by only 50%. In addition, the immobilized template was stable toward temperatures required for preparing sequencing ladders, even under cycling conditions. Standard Sanger dideoxynucleotide termination performed in a large-volume (approximately 8 microL) solid-phase reactor using the thermally stable polymerase enzymes Taq and Vent and the polymerases T7 and Bst with off-line slab gel electrophoresis and autoradiographic detection indicated that acceptable fragment generation was achieved only in the case of the thermally stable polymerases. Banding was not apparent for T7 and Bst since all reagents were inserted into the column in a single plug at the beginning of the reaction. A small volume reactor (volume approximately 62 nL) was then used to perform DNA polymerase reactions and was coupled directly to a capillary gel column for separation. The capillary reactor was placed inside a thermocycler to control the temperature during chain extension and was directly connected to the gel column via zero dead volume fused-silica connectors. The complementary DNA fragments generated (C-track only) in the reactor were denatured using heat and directly injected onto the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. Extension and single-base separation resolution of the C-track, which was directly injected onto the gel column, was estimated to be > 450 bases from the primer annealing site with plate numbers ranging from 1 x 10(6) to 2 x 10(6)/m.     

Contrasting effects of single stranded DNA binding protein on the activity of uracil DNA glycosylase from Escherichia coli towards different DNA substrates

Excision of uracil from tetraloop hairpins and single stranded ('unstructured') oligodeoxyribonucleotides by Escherichia coli uracil DNA glycosylase has been investigated. We show that, compared with a single stranded reference substrate, uracil from the first, second, third and the fourth positions of the loops is excised with highly variable efficiencies of 3.21, 0.37, 5.9 and 66.8%, respectively. More importantly, inclusion of E.coli single stranded DNA binding protein (SSB) in the reactions resulted in approximately 7-140-fold increase in the efficiency of uracil excision from the first, second or the third position in the loop but showed no significant effect on its excision from the fourth position. In contrast, the presence of SSB decreased uracil excision from the single stranded ('unstructured') substrates approximately 2-3-fold. The kinetic studies show that the increased efficiency of uracil release from the first, second and the third positions of the tetraloops is due to a combination of both the improved substrate binding and a large increase in the catalytic rates. On the other hand, the decreased efficiency of uracil release from the single stranded substrates ('unstructured') is mostly due to the lowering of the catalytic rates. Chemical probing with KMnO4showed that the presence of SSB resulted in the reduction of cleavage of the nucleotides in the vicinity of dUMP residue in single stranded substrates but their increased susceptibility in the hairpin substrates. We discuss these results to propose that excision of uracil from DNA-SSB complexes by uracil DNA glycosylase involves base flipping. The use of SSB in the various applications of uracil DNA glycosylase is also discussed.     

Investigation of species specificity using nine PCR-based human STR systems

Several eukaryotic genomes contain polymorphic markers consisting of trimeric and tetrameric short tandem repeats (STR). Recent reports have demonstrated the variability of short tandem repeat (STR) polymorphisms at a variety of loci among several human population groups. Currently, there are nine commercially available STR PCR systems from Promega Corporation that may be utilized for human identification. We report here the analysis of 23 different species DNA's using these nine STR primer systems to assess their specificity for human euchromatin. The STR systems tested include, CSF1PO, TPOX, THO1, HPRTB, FESFPS, vWF and F13A01 as single systems and as triplex systems (CSF1PO/TPOX/THO1 and HPRTB/FESFPS/vWF). There were no STR PCR products observed for seventeen of the twenty-three species regardless of the STR system. Amplified STR fragments were detected in rhesus DNA for CSF1PO, TPOX and HPRTB systems. STR PCR products were detected for human, gorilla, chimpanzee, and orangutan DNAs using eight of the nine systems. FESFPS primers did not amplify DNA fragments from any of the species tested. Most of the STR PCR products detected from primate DNAs electrophoretically migrated outside of the human allelic ladder fragments and as a result, allele designations were not possible.     

Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins

Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.     

Radiation inactivation of human prostate cancer cells: the role of apoptosis

DNA extracted from all Brucella species, reference and vaccine strains were amplified by PCR using primers specific for the genes encoding a 31-kDa Brucella protein, the heat shock proteins (DnaJ, DnaK, HtrA and GroEL) and 16S RNA. No difference was found between Brucella species and biovars with all primer pairs used, even after restriction enzyme analysis of the amplified fragments. The specificity of the amplified products was confirmed by hybridization with a digoxigenin 3'-labelled specific probe and by PCR using 98 non-Brucella micro-organisms' DNA. Only Ochrobactrum anthropi and Phyllobacterium spp. yielded a PCR product by using 31-kDa DnaK, DnaJ, GroEL and 16S RNA primers. After hybridization and restriction analysis, 16S RNA fragments of 3301 and 3331 O. anthropi strains showed a total similarity to those from Brucella. A similar result was shown with DnaJ fragments obtained with 3301 strain of O. anthropi after EcoRI digestion.     

Proximal and distal biceps tendon rupture--an indication for surgery?

To obtain genetic markers linked to a specific genetic locus, genomic subtraction with a DNA pool of backcross or F2 intercross animals with a specific genotype at the locus is known to be effective. To determine whether the pooling strategy is also effective for isolation of genetic markers linked to a quantitative phenotype that can potentially be controlled by multiple genetic loci, we tested the ability of representational difference analysis (RDA) to isolate genetic markers linked to the thymus enlargement observed in the BUF/Mna (BUF) rat. This is known to be controlled by single major and minor genes, Ten1 and Ten2, on Chromosomes (Chrs) 1 and 13, respectively, both of which have dose effects on the normal WKY/Ncj (WKY) allele. DNA from an inbred WKY rat was used as the tester, and the driver was prepared from a DNA pool of 12 (WKY x BUF)F1 x BUF backcross rats with high thymus ratios (thymus weight/body weight), expected to have dominance of the BUF allele in the responsible loci. By two RDA series with the restriction enzymes BglII and BamHI, respectively, 28 polymorphic markers were isolated, and 8 of them were shown to be linked to Ten1, and one to Ten2. One of the 8 markers linked to Ten1 demonstrated no recombination in 18 rats with high thymus ratios. RDA with a DNA pool based on a quantitative phenotype (phenotype-directed RDA) can thus be considered an efficient approach for direct isolation of polymorphic markers linked to a quantitative trait.     

Membrane electrophoresis of DNA

We have separated double stranded DNA fragments by electrophoresis using a membrane instead of a gel as separating matrix. Such a membrane, containing separated DNA fragments, is suitable for subsequent hybridization analysis. The method combines electrophoretic separation and transfer onto a solid support and therefore eliminates the need for a transfer or blotting step.