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.

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.

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.

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.

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

Rapid identification of specific TEM-type β-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 β-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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

QuantiLyse: reliable DNA amplification from single cells.

Amplification of DNA sequencesfrom single cells via PCR is increasingly used in basic research and clinical diagnostics but remains technically difficult. We have developed a cell lysis protocol that uses an optimized proteinase K solution, named QuantiLyse and permits reliable amplification from individual cells. This protocol was compared to other published methods by means of real-time PCR with molecular beacons.