Sildenafil Administration in Dogs Heterozygous for a Functional Null Mutation in Pde6a: Suppressed Rod-Mediated ERG Responses and Apparent Retinal Outer Nuclear Layer Thinning.

This study was designed to assess risk for retinal toxicity associated with administration of high-dose sildenafil citrate to dogs heterozygous for a functionally null mutation in Pde6a over a 4-month period. Three Pde6a +/- dogs were administered 14.3 mg/kg sildenafil per os and two Pde6a +/- dogs placebo once daily for 16 weeks. Three Pde6a +/+ dogs were administered sildenafil for 7 days. Ophthalmic examination, vision testing, and electroretinography (ERG) were regularly performed. At study termination, dogs were euthanized and globes collected. Retinal layer thickness and photoreceptor nuclei counts were determined from plastic sections. In both Pde6a +/- and Pde6a +/+ sildenafil-treated (ST) dogs, elevation of dark-adapted b-wave threshold and unmasking of the scotopic threshold response (STR) were observed. Sildenafil treated Pde6a +/- dogs had significantly thinner ONL (24.90 +/-1.88 µm, p = 0.004) and lower photoreceptor nuclei counts (273.6 +/- 29.3 cells/100 µm, p = 0.008) compared to measurements (35.90 +/- 1.63 µm) and counts (391.5 +/-27.0 cells/100 µm) from archived untreated Pde6a +/- dogs.

An association between systemic cyclosporine administration and development of acute bullous keratopathy in cats.

OBJECTIVE: To determine whether any association exists between the onset of feline acute bullous keratopathy (ABK) and administration of systemic corticosteroid or immunosuppressive therapy. ANIMALS STUDIED: Medical records of cats diagnosed with ABK between the years of 2000 and 2008 were retrospectively reviewed. Breed, age at diagnosis, weight, systemic disease status, eye affected, ophthalmic examination findings, systemic and topical therapy instituted, dosage and duration of therapy, visual outcome and histopathological analyses were recorded in cases meeting the inclusion criteria. RESULTS: A total of 12 cats of a surveyed population of 70 167 met the inclusion criteria with 17/24 eyes affected by ABK. Medical and/or surgical therapy was utilized for management of ABK with 13/17 eyes remaining sighted at the time of last follow-up. In a subset of cases corneal cytology, aerobic bacterial culture, FHV-1 PCR, virus isolation and/or histopathology were performed; no infectious organisms were identified. A rupture in Descemet's membrane of the cornea was identified histologically in two globes. A total of 10 of 12 cats had been previously diagnosed with ongoing systemic disease. A total of 10 of 12 cats were receiving systemic therapy, and a significant association (P < 0.001) was noted between systemic administration of corticosteroids and/or cyclosporine A and the development of ABK. A total of 8 of 10 cats were administered oral prednisolone at doses between 1-2 mg/kg every 12-24 h. A total of 5 of 8 cats receiving oral prednisolone were concurrently administered oral cyclosporine at doses of 1.5-7 mg/kg every 12-24 h. Systemic cyclosporine therapy was found to be a significant risk factor (P < 0.001) for ABK development, while systemic prednisolone was not significant (P = 0.10). CONCLUSIONS: Systemic cyclosporine administration appears to be a risk factor for development of ABK in the population of cats studied.

Low-concentration initiator primers improve the amplification of gene targets with high sequence variability.

The amplification and detection of diverse strains of an infectious virus or bacteria, or variants within a gene family is important for both clinical and basic research but can be difficult using conventional PCR. This report describes and illustrates a novel closed-tube method for amplifying and characterizing heterogeneous target sequences using members of the CTX-M beta-lactamase gene family. Different subgroups of CTX-M genes exhibit low sequence identity, but accurate and efficient detection of these variants is critical because they all confer resistance to penicillin, cefotaxime, and other antibiotics of the beta-lactam class. The method combines a single pair of "thermodynamic consensus primers" (tcPrimers) with one or more "initiator primers" (iPrimers), added at low concentration (5-10 nM). Each iPrimer improves the initial amplification of one or more variants because it has fewer mismatches to its intended target than the more abundant tcPrimers. As a result of initial amplification, each heterogeneous sequence is shifted stepwise toward a better match with the tcPrimers. As soon as the tcPrimer hybridization takes place, amplification proceeds with high efficiency. The tcPrimer pairs can be designed for symmetric PCR or for Linear-After-The-Exponential (LATE)-PCR. LATE-PCR offers the advantage of generating single-stranded DNA that can be characterized for different gene variants in the same closed tube, using low-temperature mismatch-tolerant fluorescent probes.

Rapid detection and identification of hepatitis C virus (HCV) sequences using mismatch-tolerant hybridization probes: A general method for analysis of sequence variation.

Detection and identification of highly variable viral sequences is important for tracking infectious outbreaks and determining treatment regimens using targeted drug therapy. This report describes a single tube assay that is able to distinguish extensive sequence variation in hepatitis C virus (HCV) by using mismatch tolerant probes to analyze single-stranded amplicons generated with reverse transcription linear-after-the-exponential PCR (RT-LATE-PCR). Detection and identification of sequences from the 5' non-coding region (NCR) of 31 different HCV strains was first evaluated via hybridization of two fluorescently labeled, mismatch-tolerant probes to synthetic DNA strands. The resulting data were used to calculate the ratio of fluorescent signals for the two probes over a wide temperature range as well as the melting temperature (Tm) of each probe with the targets. Although the Tm measurements alone distinguished only 5 sequences from the others, fluorescent signal ratio analysis provided a unique set of values for 27 of the 31 strains. RT-LATE-PCR was then used to amplify Armored RNA (AR) containing the 5' NCR of five different strains of HCV. Melting analysis of the resulting single-stranded DNA with the two probes distinguished all five AR sequences. This assay can be expanded to include additional gene segments, and it points the way to construction of highly informative single-tube assays for HCV and other RNA viruses.

Rapid detection of TEM-type extended-spectrum ß-lactamase (ESBL) mutations using lights-on/lights-off probes with single-stranded DNA amplification.

Rapid identification of specific TEM-type ß-lactamase genes in bacterial infections is important for determining appropriate clinical treatment. We report here the design and initial testing of a molecular diagnostic assay capable of amplifying a large segment of the blaTEM gene, as well as detecting widely spaced extended-spectrum ß-lactamase (ESBL) mutations and inhibitor-resistant TEM (IRT) mutations (eg, clavulanic acid resistance). Single-stranded DNA is generated using linear-after-the-exponential PCR (LATE-PCR) and is analyzed at the endpoint, using a set of four fluorescently labeled and four quencher-labeled probes in a single closed tube. These lights-on/lights-off probes work in concert to generate sequence-specific fluorescence contours over a temperature range from 25°C to 75°C. Mutant sequences from synthetic TEM gene variants and from TEM gene variants in bacterial strains generated large increases in fluorescent signal relative to that from the reference sequence for TEM-1. Clinical use of this convenient, single-closed-tube assay would make it possible to rapidly distinguish ESBL from non-ESBL variants and thereby to begin early treatment with suitable antibiotics.

Rapid detection of sequence variation in Clostridium difficile genes using LATE-PCR with multiple mismatch-tolerant hybridization probes.

A novel molecular assay for Clostridium difficile was developed using Linear-After-The-Exponential polymerase chain reaction (LATE-PCR). Single-stranded DNA products generated by LATE-PCR were detected and distinguished by hybridization to fluorescent mismatch-tolerant probes, as the temperature was lowered after amplification in 5(°)C intervals between 65°C and 25°C. Single-tube multiplex reactions for tcdA, tcdB, tcdC, and cdtB (binary toxin) sequences were initially optimized using synthetic targets and were subsequently done using genomic DNA; each target was detected and characterized by hybridization to one or more probes of a different fluorescent color. In the case of tcdC, three probes, each labeled with a Quasar fluorophore, hybridize to different locations with known mutations, including the deletion at nucleotide 117 in ribotype 027 strains and the premature stop codon mutation at nucleotide 184 in ribotype 078 strains, each of which is associated with hypervirulent infections. These and other tcdC mutations were distinguished from the reference sequence, as well as from each other by changes in the fluorescent contour generated from the combined Quasar-labeled probes. Specific variations in tcdA and tcdB were also identified in the multiplex assay, including those that identified strains lacking toxin A production. This single closed-tube assay generates substantially more information about virulent C. difficile than currently available commercial assays and could be further expanded to provide strain typing.

Bilateral uveal metastasis of a subcutaneous fibrosarcoma in a cat.

A 6-year-old neutered male domestic short-haired cat was presented to the Comparative Ophthalmology service at Michigan State University with a 3-week history of decreased appetite and redness of the left eye. The left forelimb had been removed 15 months previously because of the presence of a subcutaneous fibrosarcoma. In the left globe, a large iridal mass was associated with increased intraocular pressure and retinal detachment. A smaller mass involving the right iris was also present. Imaging revealed a 2-cm mass in the left caudodorsal lung lobe, and abdominal ultrasound showed multifocal bilateral renal masses. Aspirates of these masses were nondiagnostic. The left globe was removed for palliative reasons, and histopathology showed that fibrosarcoma was infiltrating the iris, choroid, and optic nerve. Despite systemic chemotherapy with doxorubicin, the animal died 4 months after initial presentation. Histopathology confirmed highly angioinvasive metastatic fibrosarcoma also in the right uveal tract, the lungs, and both kidneys.

LATE-PCR and allied technologies: real-time detection strategies for rapid, reliable diagnosis from single cells.

Accurate detection of gene sequences in single cells is the ultimate challenge of PCR sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of Linear-After-The-Exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at -different concentrations, but with novel design criteria to insure high efficiency and specificity. LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single-cell genetic diagnosis.

Pan-serotypic detection of foot-and-mouth disease virus by RT linear-after-the-exponential PCR.

A reverse transcription Linear-After-The-Exponential polymerase chain reaction (RT LATE-PCR) assay was evaluated for detection of foot-and-mouth disease virus (FMDV). This pan-serotypic assay targets highly conserved sequences within the 3D (RNA polymerase) region of the FMDV genome, and uses end-point hybridisation analysis of a single mismatch-tolerant low temperature probe to confirm the identity of the amplicons. An Armored RNA served as an internal control to validate virus negative results. The ability of the assay to identify FMDV was directly compared to a real-time RT-PCR assay routinely used by reference laboratories. The analytical sensitivity of the RT LATE-PCR assay was 10 genomic copies and the dynamic range of the test was identical to real-time RT-PCR based on decimal dilutions of an FMDV-positive sample. This pan-serotypic assay was able to detect FMDV in a broad range of clinical samples collected from field cases of FMD (n = 121), while samples of other viruses causing vesicular disease in livestock and genetic relatives of FMDV were negative. In addition to the laboratory-based utility of this diagnostic test, the RT LATE-PCR assay format has potential application for use in a portable ("point-of-care") device designed to achieve rapid detection of FMDV in the field.

Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing.

This protocol describes the design and execution of monoplex and multiplex linear-after-the-exponential (LATE)-PCR assays using a novel reagent, PrimeSafe, that suppresses all forms of mispriming. LATE-PCR is an advanced form of asymmetric amplification that uses a limiting primer and an excess primer for efficient exponential amplification of double-stranded DNA, followed by linear amplification of one strand. Each single-stranded amplicon can be quantitatively detected in real time or at end point. By separating primer annealing from product detection, LATE-PCR enables product analysis at low temperatures. Alternatively, each single strand can be sequenced by a convenient Dilute-'N'-Go procedure. Amplified samples are diluted with individual sequencing primers without the use of columns or spins. We have amplified and then sequenced 15 different single-stranded products generated in a single multiplexed LATE-PCR comprised of 15 pairs of unrelated primers. Dilute-'N'-Go dideoxy sequencing is more convenient, faster and less expensive than sequencing double-stranded amplicons generated via conventional symmetric PCR. The preparation of LATE-PCR products for Dilute-'N'-Go sequencing takes only 30 seconds.

Linear-after-the-exponential polymerase chain reaction and allied technologies. Real-time detection strategies for rapid, reliable diagnosis from single cells.

Accurate detection of gene sequences in single cells is the ultimate challenge to polymerase chain reaction (PCR) sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of linear-after-the-exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at different concentrations, but with novel design criteria to ensure high efficiency and specificity. Compared with conventional PCR, LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single cell genetic diagnosis.

Two-temperature LATE-PCR endpoint genotyping.

BACKGROUND: In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. RESULTS: Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles. CONCLUSION: SNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions.

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)-PCR.

Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but it currently is hampered by a labor-intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here linear-after-the-exponential (LATE)-PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thereby eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for single-nucleotide polymorphism genotyping applications, including an integrated single-cell-through-sequencing assay to detect a mutation at the globin IVS 110 site that frequently is responsible for beta-thalassemia.

Linear-After-The-Exponential (LATE)-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection.

Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX 5 degrees C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.

Linear-after-the-exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis.

Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)-PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80-250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CFDelta508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.

Role of the mitochondrial genome in assisted reproductive technologies and embryonic stem cell-based therapeutic cloning.

Mitochondria play a pivotal role in cellular metabolism and are important determinants of embryonic development. Mitochondrial function and biogenesis rely on an intricate coordination of regulation and expression of nuclear and mitochondrial genes. For example, several nucleus-derived transcription factors, such as mitochondrial transcription factor A, are required for mitochondrial DNA replication. Mitochondrial inheritance is strictly maternal while paternally-derived mitochondria are selectively eliminated during early embryonic cell divisions. However, there are reports from animals as well as human patients that paternal mitochondria can occasionally escape elimination, which in some cases has led to severe pathologies. The resulting existence of different mitochondrial genomes within the same cell has been termed mitochondrial heteroplasmy. The increasing use of invasive techniques in assisted reproduction in humans has raised concerns that one of the outcomes of such techniques is an increase in the incidence of mitochondrial heteroplasmy. Indeed, there is evidence that heteroplasmy is a direct consequence of ooplasm transfer, a technique that was used to 'rescue' oocytes from older women by injecting ooplasm from young oocytes. Mitochondria from donor and recipient were found in varying proportions in resulting children. Heteroplasmy is also a byproduct of nuclear transfer, as has been shown in studies on cloned sheep, cattle and monkeys. As therapeutic cloning will depend on nuclear transfer into oocytes and the subsequent generation of embryonic stem cells from resulting blastocysts, the prospect of mitochondrial heteroplasmy and its potential problems necessitate further studies in this area.

Detection of cystic fibrosis alleles from single cells using molecular beacons and a novel method of asymmetric real-time PCR.

We present a method for rapid and accurate identification of the normal and DeltaF508 alleles of the cystic fibrosis (CF) gene in single human cells that utilizes LATE (linear after the exponential)-PCR, a newly invented form of asymmetric PCR. Detection of the single-stranded amplicon is carried out in real time, using allele-specific molecular beacons. The LATE-PCR method permits controlled abrupt transition from exponential to linear amplification and thereby enhances the fluorescent signals and reduces variability between replicate samples relative to those obtained using typical real-time PCR. Of 239 single lymphoblasts generating amplification signals, 227 (95%) exhibited signals that met objective quantitative criteria required for diagnosis. Among these samples, 222 were genotyped correctly, for an assay accuracy of 98%. The small number of diagnostic errors was due to allele drop-out among heterozygous lymphoblasts, 4/119 (3.4%), and contamination among homozygous DeltaF508 lymphoblasts, 1/57 (1.8%). LATE-PCR offers a new strategy for preimplantation genetic diagnosis and other fields in which accurate quantitative detection of single copy genes is important.

Preimplantation genetic diagnosis for a couple with recurrent pregnancy loss and triploidy.

BACKGROUND: Triploidy may arise from fertilization of a mature haploid egg by two haploid sperm or by failure of meiotic divisions yielding a diploid gamete. We encountered a couple with habitual abortion, in which the last two fetuses were documented as viable triploid. METHODS: To avoid dispermic penetration and development of abnormal preembryos, insemination was done by intracytoplasmic sperm injection (ICSI) followed by fluorescence in situ hybridization (FISH) of biopsied blastomeres. RESULTS: Tests of the husband's spermatozoa by FISH, revealed that only 2-3% of the sperm were disomic for chromosomes 16, 13, 21, X, and Y. No triple disomy was detected among chromosomes 16, 13 and 21, which makes it very unlikely that triploidy resulted from diploid spermatozoa. Following a controlled ovulation induction protocol, low quality oocytes with immature cumuli were revealed. After ICSI, five eggs became two pronuclei (2PN) zygotes and none of the other eggs developed a 3PN zygote. FISH was performed on chromosomes 16 and 21 in four preembryos developed to a 6-8 cell stage. Aneuploidy or mosaicism for each of these chromosomes was detected in one preembryo and later in two disaggregated blastocysts. FISH failed in one preembryo that became atretic after biopsy. CONCLUSIONS: Although this case was unsuccessful in achieving embryo transfer and normal pregnancy, we detected many abnormal morphological features in the oocytes and chromosomal abnormalities in the cleaving preembryos. This protocol can be proposed to patients with recurrent pregnancy loss associated with chromosomal abnormalities in the fetus.

Real-time PCR with molecular beacons provides a highly accurate assay for detection of Tay-Sachs alleles in single cells.

The results presented here provide the first single-cell genetic assay for Tay-Sachs disease based on real-time PCR. Individual lymphoblasts were lysed with an optimized lysis buffer and assayed using one pair of primers that amplifies both the wild type and 1278 + TATC Tay-Sachs alleles. The resulting amplicons were detected in real time with two molecular beacons each with a different colored fluorochrome. The kinetics of amplicon accumulation generate objective criteria by which to evaluate the validity of each reaction. The assay had an overall utility of 95%, based on the detection of at least one signal in 235 of the 248 attempted tests and an efficiency of 97%, as 7 of the 235 samples were excluded from further analysis for objective quantitative reasons. The accuracy of the assay was 99.1%, because 228 of 230 samples gave signals consistent with the genotype of the cells. Only two of the 135 heterozygous samples were allele drop-outs, a rate far lower than previously reported for single-cell Tay-Sachs assays using conventional methods of PCR.