Detection of asymmetric PCR products in homogeneous solution by fluorescence correlation spectroscopy

The yield of the double-stranded DNA product (500 bp) of asymmetric PCR with a rhodamine-labeled primer (Rho-primer) was determined in a homogeneous solution using fluorescence correlation spectroscopy (FCS). FCS provides the average number of molecules in a focused volume and the diffusion constant that relates the molecular weight. Since FCS measures the fluctuation of fluorescence intensity in a very small sample volume, the reaction mixture was directly placed on the FCS optical field without any purification procedure after amplification. The result of changing the initial number of templates suggested that elongation of the Rho-primer could be detected by FCS in a PCR mixture containing a single copy of the target gene in the initial condition. Possible scientific applications and perspectives of the proposed approach are discussed.     

Rescue thrombolysis with repeated doses of streptokinase in acute myocardial infarct. A preliminary report

A study was made on various methodological aspects of fluorescence image cytometry (FICM) for measurement of nuclear DNA content by using CCD cameras attached to an epifluorescence microscope. Cell nuclei of paraffin-embedded specimens from mouse tissues and human prostate carcinomas were isolated and stained with 4'-6-diamidino-2-phenylindole (DAPI). We found that fluorescence fading, lamp stability, and the homogeneity of the illumination can easily be controlled. A camera with a signal-to-noise ratio of 53 dB gave a slightly more precise measurement than did a 46-dB camera. The linearity of the analysis results was very good. The coefficient of variation of mouse kidney standard cells in the DNA histograms was about 5% and 7.4% in histograms of prostate carcinoma biopsies. Stained cell nuclei can be stored for long periods at -20 degrees C without impairment of quality. Comparative measurements of ploidy by FICM and flow cytometry confirmed the accuracy of the FICM analyses. Thus, FICM appears to be an easy method for quantifying the DNA content of visually inspected cell nuclei in surgical pathology.     

Conjugate imagery in the automated reproduction of three dimensional coordinates from two dimensional coordinate data

The three-dimensional video analysis of human motion commonly utilises automated image processing and digitisation processes to produce real-time unidentified two-dimensional coordinate data of segmental markers. In what can be a time-consuming process the two-dimensional data are then identified and tracked to produce three-dimensional coordinates. This paper presents an approach to the automated reproduction of three-dimensional coordinates from two-dimensional coordinates data. Conjugate imaging techniques were utilised in the development of four criterion measures for determining the validity of conjugate (corresponding) image points. An algorithm based on the criterion measures was then developed for the automated reproduction of three dimensional coordinates from camera image coordinate data. The algorithm was tested with a 55 point marker system viewed in four video cameras (digitisation error approx. 0.2%, lab point separation > or = 6 cm). The success of the algorithm was dependent on the closeness of markers, the accuracy of the photogrammetric system, and the number of markers visible in two camera images. The present research has developed techniques based on conjugate imagery for the automated reproduction of three-dimensional coordinates from two-dimensional data, and provided a bases for further development of automated three-dimensional tracking.     

Fucosyltransferase activities in human pancreatic tissue: comparative study between cancer tissues and established tumoral cell lines

This review focuses on polymerase chain reaction (PCR) sexing of bovine embryos in commercial situations with emphasis on new developments. Simplifications of the biopsy technique is one of the major simplifications over the last few years. The stabilization of the embryo by means of protein-free medium or scratches produced on the bottom of the Petri dish makes it possible to perform a biopsy with a single microinstrument. The traditional PCR sexing approach utilizes electrophoresis, which involves the risk of deoxyribonucleic acid (DNA) contamination of subsequent assays. Such contamination, resulting in females misdiagnosed as males, is avoided efficiently by using a non-electrophoretic method in which the sex is determined based on fluorescence of unopened tubes. However, female samples cannot be distinguished from blank samples in the non-electrophoretic assay, which thus relies on accurate transfer of biopsy into tubes. Nevertheless, an accuracy of about 95% can be reached with both approaches. High pregnancy rates (50-70%) can be reached with biopsied Grade 1 embryos, but there is evidence that pregnancy rates with Grade 2 embryos is 15-20% lower. Recent data indicate that pregnancy rates of 50% can be achieved with frozen-thawed biopsied Grade 1 embryos. In conclusion, recent developments in biopsy techniques, detection systems and freezing should increase interest in PCR sexing.     

Genetic diagnoses of Chlamydia trachomatis--DNA probe and PCR method

Chlamydial infections such as male urethritis or female cervicitis, although not receiving as much attention as the AIDS epidemic, are on the increase around the world as sexually-transmitted diseases (STD). A number of diagnostic methods i.e., direct fluorescence antibody method and enzyme immunoassay (EIA) methods, have been developed for detecting Chlamydia trachomatis (C. trachomatis) infection, but they each have a defect in terms of sensitivity or specificity. Then, to overcome these disadvantages, the genetic diagnostic method such as DNA-RNA hybridization (DNA probe) method, PCR method and LCR method have been clinically introduced. In in vitro studies, the PCR method has higher sensitivity than the EIA method or DNA probe method. All genetic diagnostic methods have specificity superior over the EIA method. In clinical studies, the PCR method was the most useful for detection of C. trachomatis from various infectious lesions.     

Preparation of uniformly isotope-labeled DNA oligonucleotides for NMR spectroscopy

Two methods for the large scale preparation of uniformly isotope-labeled DNA for NMR studies have been developed. The first method comprises the growth of a suitable plasmid harboring multiple copies of the desired oligonucleotide in a medium based on 15N and 13C nutrients. The second method uses a polymerase chain reaction (PCR)-based approach with 15N- and/or 13C-labeled deoxynucleoside triphosphates. The novelty of our PCR strategy over existing ones is that the primer and template are the identical molecule, resulting in an exponential growth in the length of the double strand that contains tandem repeats of the target DNA sequence. This novel PCR approach, which we have termed ESRA for endonuclease-sensitive repeat amplification, is easy to use, results in high yields, and can be accomplished at low costs. The utility of both methods is demonstrated for the preparation of a double-stranded 21-mer uniformly labeled with 15N and a double-stranded 17-mer DNA uniformly labeled with 15N and 13C.     

An Optical Approach to Structural Displacement Measurement and Its Application

A number of optical devices are commercially available now for measurement. Some of them, such as laser devices and still and video cameras with high resolution, may be used effectively and efficiently for measuring structural displacement. This paper presents an approach to this type of application using a charge-coupled-device camera. It can acquire digitized images for low cost, to be used to identify structural displacement via digital signal processing. It is shown that this approach’s resolution for point measurement is comparable with traditional sensors such as dial gages. Furthermore, it offers a new capability of displacement measurement for a large number of points on a structure, and it can provide spatially intensive displacement data. This kind of data may be used for structural damage detection and health monitoring, as suggested and demonstrated herein.     

PCR amplification of murine immunoglobulin germline V genes: strategies for minimization of recombination artefacts

Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the 'plateau phase' of the reaction was reached. Recombination events were located throughout the approximately 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.     

Use of polymerase chain reaction to assess efficacy of leprosy chemotherapy

The assessment of chemotherapy efficacy in leprosy is difficult, since the only reliable method for determining whether the causative organism, Mycobacterium leprae, is viable depends on its growth in mouse foot pads. In an attempt to replace this expensive, time-consuming test, methods based on the polymerase chain reaction (PCR) have been developed. These methods depend on detection of DNA, which is more susceptible to degradation on cell death than are other cell components, so should be a more accurate indicator of viability. We have used a specific PCR assay to detect M leprae DNA in skin biopsy samples from leprosy patients. By use of limiting dilution PCR (LD-PCR), the concentration of M leprae DNA in the original sample could be measured. The DNA concentration was more closely correlated with the morphological index (derived from a staining technique that distinguishes morphologically intact and damaged bacteria) than with the number of bacteria visible (bacterial index, BI, which counts both alive and dead bacteria). In a longitudinal study of multibacillary patients on multi-drug therapy, skin biopsy samples were collected before treatment and 3, 6, 12, and 24 months after the start of therapy. While the BI showed little or no change during treatment, the number of genomes detected by PCR fell sharply, in parallel with the MI. We propose that PCR can be used as a rapid measure of M leprae viability and that this approach can be used for monitoring individual leprosy patients and for assessment of existing and new regimens. The method may be applicable to other infectious diseases in which culture of the causative organism is slow or impossible.     

Fluorescence correlation spectroscopy of enzymatic DNA polymerization

We show that fluorescence correlation spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.     

Genotypic selection methods for the direct analysis of point mutations

Genotypic selection enriches a particular DNA sequence relative to another closely-related DNA sequence based only on a change of one or a few bases. This review is a survey of the genotypic selection methods that have the sensitivity to detect rare point mutations. These methods are primarily being used to study mutations caused by environmental mutagens; however, the ability to detect and measure very minor DNA sequence populations is likely to further research efforts in many fields. The approaches for allele-selection have intrinsic strengths and weaknesses, and vary greatly in sensitivity. The most sensitive method is Restriction Fragment Length Polymorphism/Polymerase Chain Reaction (RFLP/PCR) by which mutant fractions as low as 1 mutant allele in 10(8) wild-type alleles can be detected. The RFLP/PCR approach is presented as a prototype genotypic selection method. Genotypic selection methods are categorized in terms of those that (1) selectively destroy the abundant or wild-type allele, (2) selectively amplify the rare or mutant allele, or (3) spatially separate the alleles. Issues relevant to the further development of genotypic selection methods include initial DNA pool size, strategies to eliminate the bulk of extraneous DNA, the use of an internal copy number standard in quantitative PCR, the fidelity of thermostable DNA polymerases, and the effective use of PCR in linking two or more genotypic selection techniques. We conclude that proficient genotypic selection requires more than one allele-enrichment technique with at least one of these preceding a high-fidelity PCR amplification step.     

Late effects of radiotherapy in children

PURPOSE: To examine benzoporphyrin derivative angiography as a modality for studying photosensitizer biodistribution in experimental choroidal melanomas. METHODS: A liposomal preparation of benzoporphyrin derivative was used in this study. Digital benzoporphyrin derivative angiograms were performed in 10 rabbits (six for experimental choroidal melanomas, two for normal choroids, and two for irides) using a Topcon ImageNet H1024 digital imaging system, a Kodak Megaplus video camera, and a Topcon TRC-50-VT fundus camera. Only one eye from each rabbit was used. Filters specifically designed for benzoporphyrin derivative (peak absorption at 580 nm and peak emission at 695 nm) were used. Benzoporphyrin derivative (1 mg/kg) was injected into an ear vein while images of tumor, normal choroid, or iris were being obtained. Follow-up images were obtained during the first 3 hours and at 24 hours after injection. Fluorescence microscopy was performed in all 10 rabbits using 1 mg/kg of benzoporphyrin derivative. Tumor-bearing eyes were enucleated at the same time points that angiograms were performed, and the two sets of results were compared for maximum dye accumulation. RESULTS: Digital angiography demonstrated that maximal benzoporphyrin derivative fluorescence occurred in tumors 15 to 45 minutes after injection. Fluorescence photometry corroborated these results. CONCLUSION: Photosensitizer angiography is a valid modality for determining the optimum treatment time for photodynamic therapy.     

Analysis of single-strand conformation polymorphisms by capillary electrophoresis with laser induced fluorescence detection

Detection of point mutations in genomic DNA is important for diagnosis of inherited characteristics and genetic diseases. A point mutation in a specific region of DNA amplified by polymerase chain reaction (PCR) can be detected with single-strand conformation polymorphism (SSCP) analysis. Analysis of SSCP by laser-induced fluorescence capillary electrophoresis in entangled polymer solution (CE-LIF) has been developed in the present paper. K-ras genes including seven mutations were amplified with primer labeled with Texas Red at its 5' end. The labeled PCR products were dissociated to single strands by heating and separated with capillary gel electrophoresis and He-Ne laser-excited fluorescence detection. Our results suggest that all fragments having normal (Gly) and mutated (Ala, Arg, Cys, Ser, Val, Asp) sequences at codon 12 can be distinguished. Analysis of SSCPs with CE-LIF is well suited for clinical analysis of SSCPs because of its high sensitivity, resolution, reproducibility and speed.     

Quantitative analysis of polymerase chain reaction using anisotropy ratio and relative hydrodynamic volume of fluorescence polarization method

The method based on the combination of polymerase chain reaction (PCR) and fluorescence polarization is presented. A targeted DNA was amplified with a 5'-fluorescein labeled primer, using a 256 bp DNA fragment of stx2 gene in Escherichia coli O157:H7 (188-443 bp) as a template. The fluorescence anisotropy of the 5'-fluorescein labeled primer increased upon the polymerization through Taq polymerase. The conversion of primer to PCR product was quantitatively monitored by anisotropy ratio and relative hydrodynamic volume. This system was also applied to the determination of E.coli O157:H7.     

Quantitation of human cytomegalovirus glycoprotein H gene in cells using competitive PCR and a rapid fluorescence-based detection system

A technique is described for quantitation of the human cytomegalovirus (HCMV) glycoprotein H (gH) gene in cells using a quantitative-competitive polymerase chain reaction (QC-PCR). Two recombinant DNA molecules, differing in size due to a 92-bp deletion within the HCMV gH sequence, were used in co-amplification studies to construct a standard curve from which the copy number of the gH gene present in clinical samples could be interpolated. The use of primers labeled with a fluorescent dye allowed direct detection of the amplified products by measuring the amount of fluorescence emitted by each specific PCR fragment with an automated DNA sequencer coupled to a software program. This system was validated subsequently using bronchoalveolar lavage cells obtained from immunocompromised patients and found to be highly sensitive and reproducible over a range of 5-50,000 HCMV gH copies. This rapid procedure could easily be applied to study the pathogenesis of HCMV infection, identify the patients at high risk of developing HCMV disease, and monitor the effects of antiviral therapy at the molecular level.     

Short-term stability of HIV provirus levels in the peripheral blood of HIV-infected individuals

Changes in viral load have been reported to reflect disease progression or response to therapy; however, the stability of HIV DNA levels in HIV-infected individuals has not been extensively studied. Cellular HIV DNA levels in infected individuals were evaluated over a short time period to determine degree of variability as well as any correlation with other measurements of virus load or immune status. Peripheral blood mononuclear cells (PBMC) were obtained several times over 1 month from 32 asymptomatic or symptomatic non-AIDS, HIV-infected individuals currently on AZT therapy. PCR amplification of the HIV gag region was performed with DNA from PBMC lysates and the PCR amplified products quantitated by liquid phase hybridization. HIV DNA levels in the majority of the patients were relatively stable, with 26 of 32 persons having less than threefold change. Changes over the study period were both positive and negative, and the median change in HIV DNA levels was 68.6%. These changes were found to positively correlate with fluctuations in plasma p24 levels. In contrast, no correlations were found with other measurements of immune system activity, including changes in CD4 number, CD4 percent, and beta 2-microglobulin when compared with provirus changes. This study shows that levels of HIV DNA can be relatively stable over short periods in most non-AIDS, HIV-infected persons.     

Construction of two near-kilobase resolution restriction maps of the 5' regulatory region of the human apolipoprotein B gene by quantitative DNA fiber mapping (QDFM)

Quantitative DNA fiber mapping (QDFM) is a high-resolution technique for physical mapping of DNA. The method is based on hybridization of fluorescently labeled DNA probes to individual DNA molecules stretched on a chemically modified glass surface. We now demonstrate and validate a rapid QDFM-based approach for the mapping of multiple restriction sites and precise localization of restriction fragments in large genomic clones. Restriction fragments of a 70-kb P1 clone (P1-70) containing the 5' region of the human apolipo-protein B gene (APOB) were subcloned and mapped along straightened P1-70 DNA molecules. Multicolor fluorescence in situ hybridization (FISH) and digital image analysis allowed us to rapidly position 29 restriction fragments, ranging in size from 0.5 kb to 8 kb, and to map 43 restriction sites. The restriction map obtained by QDFM was in excellent agreement with information obtained by RecA-assisted restriction endonuclease (RARE) cleavage, long-range PCR, and DNA sequence analyses of the P1-70 clone. These data demonstrate that QDFM is a rapid, reliable method for detailed restriction site-mapping of large DNA clones.     

A new reconstructive procedure, interposition of a jejunal pouch after proximal gastrectomy

Cough is a common symptom of pulmonary diseases. For a number of reasons it would be of interest to have information about the frequency of coughs over a given period of time. So far, the cough recorders which are available are either too expensive or unwieldy. Hence, we developed a cough recorder linked to a portable, commercially available actigraph (about the size of a pack of cigarettes) that records coughing as an acoustic signal and ventral thorax movement. The signals are filtered via a band pass and sampled by a peak detektor with different time constants to separate the impulse character of the cough signal from the background noise level. The cough recorder registers coughing cumulatively over a period of one minute and has a storage time of one week. Since the acoustic signals are essential for the interpretation of the recordings, the analogue circuit was subjected to a separate validation programme. For this purpose, the distinction between active coughs of 10 volunteers (total number of coughs 550) and background noises (male and female voices and other defined noises, total number of noises 336) was tested. The complete assembly was then tested over night on 7 hospitalised patients with chronic cough. An infrared video camera system was used to make a reference recording of the overnight coughing. The results show that nearly every cough of the 10 volunteers was recorded (r = 0.99). 97.1% of the background noise was correctly interpreted. The complete recorder assembly correctly recorded 98.9% of the coughs (total 870) in the 7 patients. 4.8% of the background noise was erroneously registered as coughing. Summing up, it can be said that the portable cough recorder affords accurate recording of coughing over a period of one week, correctly distinguishing coughing from background noise.     

Detection of HIV1 DNA in biological samples by an homogeneous assay: fluorescence measurement of double-stranded RNA synthesized from amplified DNA

A nonisotopic homogeneous detection of nucleic acid sequences after amplification is described. We show that a DNA fragment bearing T7 RNA polymerase promoters on each extremity is able to be transcribed in two complementary RNAs, leading to a high yield direct synthesis of double-stranded RNA (dsRNA). Thus, this dsRNA can be easily detected and quantified in solution by fluorescence in the presence of propidium iodide. This reaction, used as a postamplification step, has been associated with a nested polymerase chain reaction (PCR); the second PCR round allowing the incorporation of the T7 promoters. This leads to a very efficient homogeneous assay. The fluorescence signal is proportional to the concentration of PCR product and is highly specific. This method can be easily carried out with currently available reagents and with unsophisticated instrumentation. This homogeneous procedure has been evaluated for the detection of HIV1 in blood samples; the sensitivity and the specificity appear to be equivalent to that of the radioactive method.     

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.