Threat Agent Detection Impetus For Handheld Nucleic Acid Analyzer Development

Successful performance of PCR obviously depends on the ability of the thermal cycling platform to maintain accurate gradations of temperature in the manifold or block containing the reaction tubes. The heating and cooling apparatus necessary to provide these temperature changes places constraints on the size and weight of the thermal cycler. ''First-generation'' thermal cyclers, such as the Perkin Elmer 480 model instrument, while reasonably light in weight and small in footprint, were not...

Familial Adenomatous Polyposis Syndrome

Familial adenomatous polyposis syndrome is characterized by accelerated tumorigenesis from normal mucosa to polyps. It occurs in 1 8000 to 1 13,000 live births and shows no racial, gender, or geographical predilection. It has autosomal dominant inheritance with 100 pene-trance, and if left untreated, patients affected with FAP will inevitably develop colorectal cancer, typically by the age of 35-40 (mean age 39 years). 1 It accounts for about 1 of all colorectal cancers. Approximately 15 , 75 ,...

G390v F418s

Fig. 1 Mutations found in the serum cholinesterase gene. (Mutations F28I, N96Y, 317-318insA, R386C, R424X, and E460K come from Ref. 7 . All of the others come from Ref. 2 .) serum cholinesterase is 2.2 and low FN or DN FN is 1.1 in these peoples. 1 These values show that polymorphism is very common in normal populations. Distinct ethnic differences in genetic mutations of the human BChE gene have been reported. The P100S, L330I, G365R, and R515C were only found in Japanese, whereas A (G70D),...

Visceral Signs And Symptoms

Splenomegaly exists in all patients, and is typically associated with hypersplenism, early satiety, and, in children, growth (height) retardation. Anemia and throm-bocytopenia, resulting in fatigue and bleeding tendency, respectively, are among the earliest presenting signs and most prominent features of Gaucher disease. In almost all symptomatic patients, there is some degree of hepatomegaly but only rarely are liver function tests abnormal. Bone involvement is among the more variable and...

Other Mutations In The 12S rRna Gene

Deletion of a T at position 961 and insertion of varying numbers of G were observed in a patient during a search in the mtDNA in 35 sporadic Chinese cases with AID. 30 The same mutation was not detected in 799 control samples. 30 This mutation was later found in an Italian family, in which five maternal relatives became deaf after aminoglycoside exposure. 31 However, in a recent study, 961delTinsC(n) was detected in 7 of 1173 anonymized blood samples in the United States. 24 This result...

Clinical Description

NF1 is a progressive multisystem disorder, which includes some features that occur early in life and others that do not appear until later. It displays a wide range of variable expression, including differences within the same family. The hallmark feature is the neurofibroma, a benign tumor that arises from the nerve sheath. 1 There are two major types of neurofibromas focal growths and plexiform neurofibromas. Focal neurofibromas arise as discrete nodules at some point along a nerve. They may...

Hbv Dna Quantitation

For quantitation of HBV DNA, a commercial third-generation assay is available from Bayer Diagnostics (VERSANT HBV DNA 3.0). Results of a multicenter evaluation showed that the specificity of this test was 99.3 using 600 HBV seronegative samples at four different sites. Quantitation was possible between 2 x 103 and 1 x 108 copies HBV DNA mL and the assay proved to be linear throughout its entire dynamic range. Mean coefficients of variations of 7.5 and 13.6 were established for intra- and...

Genetic Alterations In

Familial adenomatous polyposis is caused by inheritance of germline mutation in the APC gene (chromosomal locus 5q21-q22). Familial adenomatous polyposis is an autosomal dominant disease with extremely high penetrance, as 95 of individuals with FAP have polyps by age 35. Approximately 20-25 of individuals with FAP have the altered gene as a result of a de novo gene muta-tion 8 and therefore will not have an affected parent. The APC gene is a ''gatekeeper'' tumor suppressor gene encoding a large...

Clinical Diagnostic Criteria for Cowden Syndrome

The International CS consortium has developed diagnostic criteria for this condition. 3,11 The criterion is less sensitive in children and young adults as findings appear generally in the third decade. Studies of patients who only partially meet these criteria, 'CS-like', or who have multiple primary cancers, have yielded very few germline PTEN mutations, thus providing indirect evidence of the usefulness of these criteria. 12,13 Clinical Management of Cowden Syndrome Clinical management of a...

Gsd Type Xi Hepatic Glycogenoses With Renal Fanconibickel Syndrome

This rare GSD is caused by defects in the facilitative glucose transporter 2 (GLUT-2), which transports glucose in and out of hepatocytes, pancreatic cells, and the basolateral membranes of intestinal and renal epithelial cells. 30 The disease is characterized by proximal renal tubular dysfunction, impaired glucose and galactose utilization, and accumulation of glycogen in liver and kidney. The affected child presents in the first year of life with failure to thrive, rickets, hepato- and...

Gsd Type Vi Liver Phosphorylase Hers Disease

The enzyme deficient in type VI GSD is liver-specific phosphorylase. It is relatively rare and a benign form of GSD with no heart and skeletal muscle involvement. Most patients with GSD VI disease present with hepatomegaly and growth retardation. Hypoglycemia, hyperlipidemia, and hyperketosis, if present, are usually mild. The hepatomegaly improves and disappears around puberty. Diagnosis rests on enzyme analysis of the liver biopsy. GSD VI is autosomal recessive. Liver phosphorylase gene...

Procedures For Msms Screening

Specimen preparation for MS-MS screening requires an extraction and derivatization process, 3 which is performed using 96-well polypropylene microplates in routine analysis. 4-6 One 3.2-mm disk of dried blood spot is punched out into the individual wells of a microplate. To the disk in each well, a methanol solution containing known concentrations of stable isotope-labeled standards is added. The microplate is covered and the samples are mixed. The extract is transferred to a second microplate...

Oxidative Stress Related Disorders

Oxidative stress is implicated in the inflammatory demy-elination that characterizes multiple sclerosis suggesting GST polymorphisms may be associated with disability. In 177 patients with disease duration over 10 years, GSTM3 AA (OR 2.4) and homozygosity for both GSTM1*0 and GSTP1*Ile105-encoding allele (OR 5.0) were linked with severe disability suggesting that long-term prognosis in MS is influenced by GST-mediated ability to remove toxic products of oxidative stress. 1 Exposure to...

Principles Of Qfish

As stated above, Q-FISH requires the use of fluorescently labeled telomeric PNA probes. The most commonly used probe is Cy3-labeled (CCCTAA)3. Following hybridization of this probe with the metaphase chromosome preparations, high stringency washes are performed to eliminate background fluorescence that may interfere with the quantitation of telomere fluorescence. Separate images of metaphase chromosomes and telomeres are then acquired using appropriate image-acquisition software packages and...

Features Of Fraxa And Fraxe

Clinical Phenotype, Genetics, and Biochemistry The fragile X syndrome affects males to a greater extent than females in males, learning and social deficits are moderate to severe, with social impairments usually preventing them from fathering offspring although sperm production is thought to be unaffected. In adult males, visible signs of the condition often include a large head, long face, large ears, and macroorchidism however, in children, the phenotype is likely to be restricted to...

Clinical Utility Of Fish Detection Of Recurring Numerical And Structural Chromosomal Abnormalities In Acute Myeloid

The convention established by the Fourth International Workshop on Chromosomes in Leukemia 32 serves as the basis for the definition of a clonal abnormality. In clinical cytogenetics, the presence of two or more trisomic or structurally rearranged cells, or three or more monosomic cells, is considered clonal. 4 Following this criterion, a number of recurring clonal cytogenetic abnormalities have been identified in AML based on G-banded studies alone. Selected chromosomal abnormalities found in...

Microarrays For Diagnostic Purposes

The development of high-density microarrays offers an enormous potential for diagnostics. Where standard pathological and molecular techniques cannot distinguish between tumor subclasses, molecular profiles are now able to predict clinical behavior. 1-3 The Dutch Cancer Institute has developed a signature set of 70 genes that can predict the 5-year survival chance of breast cancer patients and is planning to use this information to decide whether or not to start chemotherapy treatment. 2 A set...

Development Of Dnabased Diagnostic Tests For O Volvulus

Development of DNA-based detection methods for O. volvulus began in the 1980s before the development of the polymerase chain reaction (PCR). Using conventional screening approaches, several DNA probes were identified that exhibited varying specificities. 1 Some were specific for parasites of the genus Onchocerca, 2 while others were specific for O. volvulus. 3,4 One probe (pFS-1) was developed that was specific for parasites endemic to the rain forests of West Africa. 5 DNA sequence analysis...

The Amplicor HCV Monitor 20 and Cobas Amplicor HCV Monitor 20 Test Roche Diagnostic Systems

Based on reverse transcription polymerase chain reaction (RT-PCR), the Amplicor HCV Monitor test (Roche Diagnostic Systems) was developed. 5 Recently, an improved version 2.0 was introduced, which achieved an equivalent quantitation of each genotype over the quantitative range (5 x 102 to 5 x 105 copies of RNA mL). 6,7 HCV RNA is extracted from plasma by chaotropic salt and is then precipitated by isopropanol. Both RT and PCR of RNA are accomplished in one tube using the recombinant...

Management

There is currently no cure or prevention for AD because its basis are still not well understood. Pharmacological agents and psychosocial intervention may help to manage each symptom and provide temporary improvement in cognitive functioning for some patients. Cholinesterase inhibitors such as Tacrine (with hepatotoxic effects), Aricept, Exelon, and Remynil are the most used. Nonsteroidal antiinflammatory drugs (NSAIDs), estrogen replacement therapy (ERT), and lipid-lowering agents are also...

Substrate Reduction Therapy

Oral substrate reduction therapy (SRT) involves attenuation of the rate of synthesis to achieve a balance with reduced endogenous glucocerebrosidase. A pivotal trial of SRT was carried out in 28 adult patients (from four centers Cambridge, UK Amsterdam Prague and Jerusalem) naive to enzyme therapy, with mild to moderate type I Gaucher disease, who were unable or unwilling to receive enzyme treatment. There was significant reduction in spleen and liver, although amelioration of anemia and...

MagNA Pure Lc Instrument

Magnetism is the underlying principle of the automated nucleic acid isolation performed by the MagNA Pure system (Roche Diagnostics, Mannheim, Germany). The instrument has a completely closed housing as well as an automatic clot and tip loss detection. No filtration, centrifugation, or vacuum pumps are necessary, which minimizes the risk of cross-contamination. Up to 32 nucleic acid isolations (the eight-nozzle pipette head allows a variable number of samples from 1 to 32 per run) can be...

References

Barker, P.E. Srivastava, S. Atha, D.H. Validation of telomerase activity for cancer early detection TRAP PCR-CE and hTERT mRNA quantification assay for high-throughput screening of tumor cells. J. Mol. Diagnostics 2004. 6, 157-165. 2. O'Connell, C.D. Tulley, L. Devaney, J. Marino, M.A. Jakupciak, J.P. Atha, D.H. Renewable standard reference material for the detection of TP53 mutations. Mol. Diagn. 2003, 7, 85-97. 3. Duewer, D.L. Currie, L.A. Reeder, D.J. Leigh, S.D....

Human Papillomavirus Types

Human papillomaviruses (HPVs) are associated with various benign and malignant epithelial proliferative diseases. Over 100 genotypes of HPV are recognized. An HPV isolate is described as a novel type if the nucleotide sequence of its E6, E7, and L1 genes differs more than 10 from that of any other HPV type. Based on tropism, a distinction can be made between cutaneous HPV types that infect the epidermis and mucosal HPV types that infect the epithelia of the anogenital or the aerodigestive...

Performance Characteristics

The performance requirements for a good molecular typing system include a high index of strain discrimination (> 0.95) and assay reproducibility, and the ability to provide results for a diverse group of microorganisms. 10 Pulsed field gel electrophoresis technology satisfies these criteria and therefore is the most frequently used molecular typing method in molecular epidemiology. The PFGE band patterns represent the entire microbial chromosome profile (> 90 ). 11 All manipulations for DNA...

COBAS Ampli Prep

The COBAS AmpliPrep robotic workstation (Roche Diagnostics, Mannheim, Germany) is a fully automated system which isolates DNA and RNA targets. It is based on silico-coated glass particles which bind the extracted nucleic acids. Internal controls allow target recovery monitoring. Up to 72 samples (three racks of 24 specimens each) can be loaded on the system simultaneously. Reagents are delivered in sealed, bar-coded, ready-to-use cassettes. The pipetting integrity check ensures the accurate...

Conclusion Genomics And Vaccination

Despite the advances associated with molecular techniques, meningococcal disease remains a globally significant health problem, and the prospect of comprehensive vaccination remains elusive. Whereas vaccines based on the capsular polysaccharide are either licensed or under development against meningococci associated with serogroups A, C, and Y, as well as W-135, attempts to develop a polysaccharide-based serogroup B vaccine have proven unsuccessful. This fact is especially relevant as much of...

PCRRestriction Fragment Length Polymorphism RFLP

The most commonly used method to confirm genetically the diagnosis of SMA is a qualitative PCR-RFLP assay to detect the homozygous absence of SMN1. 22 The PCR-RFLP assay takes advantage of the base differences in exons 7 and 8 to distinguish SMN1 from SMN2. The restriction endonuclease Dral digests only the SMN2 exon 7 PCR products because of a restriction site generated by a mismatched reverse primer. 22 Another method introduces HinfI control restriction sites in both the SMN1 and the SMN2...

Molecular Genetics

The first identified melanoma susceptibility gene, located on chromosome 9p21-p13, has been known by many different names MTS1, INK4A, CDKN2. The currently accepted gene nomenclature is CDKN2A, which stands for cyclin-dependent kinase inhibitor 2A. This locus was mapped by linkage analysis in families in which melanoma was considered the sole phenotype, rather than including a nevus phenotype as well. 13 By using different first exons, exon 1a and 1p, respectively, CDKN2A encodes for two...

Detection Of cKit D816V In Various Subtypes Of Mastocytosis

Detection of the D816V mutation in an extracutaneous organ is a minor criterion for the diagnosis of SM SM criterion . 3 In the presence of other i.e., one major or two minor SM criteria, the final diagnosis SM can be established 3- Table 1 . Patients with cutaneous masto-cytosis CM may also exhibit D816V in their skin or even in BM , but other criteria for SM are not fulfilled. In the diagnostic workup of suspected SM, analysis for the D816V mutation is routinely performed on BM and PB...

Differential Diagnosis

Most clinicians still rely on a set of criteria originally formulated by Kimonis et al. 1 Table 1 shows a current list of qualifying criteria. It is worth noting that a number of other related but apparently distinct disorders are recognized. Rombo syndrome 2 is a familial disorder with vermiculate atrophoderma, milia, hypotrichosis, trichoep-itheliomas, basal cell carcinomas, and peripheral vasodi-lation with cyanosis, whereas Bazex-Dupre-Christol syndrome 3 combines major characteristics of...

Clinical Diagnosis And Genetic Testing

Diagnosis of this syndrome requires evaluating a combination of clinical criteria including a thorough family history, histological tumor characteristics, and consideration of genetic testing. The International Collaborative group for HNPCC initially developed the Amsterdam Criteria to help identify potential HNPCC patients. 9 However, 60 of families with a known germline mutation do not meet the criteria for the disease, 10 and only 40-80 of patients meeting the criteria have an identified...

Genetic Testing For Stk11

Genetic testing for germline STK11 mutations is currently offered at four clinical laboratories.1-29-1 DNA specimens isolated from peripheral blood or the buccal mucosa can be tested utilizing either genetic sequencing of the entire coding region, mutation scanning e.g., SSCP, DHLPC, DGGE, etc. , or mutation analysis i.e., assessment for specific mutations . The variability in the types of STK11 mutations identified in PJS families to date suggests that no single currently available mutation...

Molecular Diagnosis Of Alphathalassemia

The presence of alpha-thalassemia, suspected on the basis of hematological and or clinical findings i.e., micro-cytosis with normal HbA2 and F and normal serum iron status in carriers microcytic and hypocromic hemolytic anemia with RBC inclusion bodies and a fast-moving band HbH at the hemoglobin electrophoresis presence of Hb Bart's in the newborn , should be confirmed, if requested, by globin gene synthesis or even better by globin gene DNA analysis. The exact definition of the genotype with...

Info

Forms of the disease presenting with different ages of onset and clinical severity have been identified.1-12'13-1 The infantile form presents with progressive hypertrophic cardiomyopathy, generalized hypotonia with muscle wasting' hepatomegaly' and macroglossia' followed by a rapid, progressive course. Death usually occurs before the second year of life as a result of cardiorespiratory failure. The juvenile form presents in early childhood, with progressive muscle weakness including the...

Types Of Gaucher Disease

Gaucher disease has classically been divided into three clinical types based on the absence or presence of neurological involvement. Type I is defined by the absence of neurological involvement and, usually, the presence of the N370S 1226G mutation on at least one allele. Although pan- MOLECULAR, GENETIC, AND ENZYMOLOGICAL ASPECTS The genomic area near the p-glucocerebrosidase locus on human chromosome 1q21 includes its pseudogene and several other gene loci. Enzyme and mRNA activities vary...

Advantages And Disadvantages

One advantage of NASBA and TMA compared to PCR is that they are continuous, isothermal processes that do not require a thermocycler. The constant temperature maintained throughout the amplification reaction allows each step of the reaction to proceed as soon as an amplification intermediate becomes available. Thus, the exponential kinetic of the amplification process, which is caused by multiple transcription of RNA copies from a given DNA product, is intrinsically more efficient than DNA...

Human Platelet Antigens Nomenclature

Historically, multiple names existed for the human platelet antigens. In 1990, the Platelet Serology Working Party of the International Society of Blood Transfusion ISBT and the International Committee for Standardization in Haematology ICSH suggested a numerical terminology which has been widely accepted. 1 The term HPA was introduced to indicate human platelet antigen. By convention, ''system'' is assigned to an HPA where both SNP and alloantibodies have been identified. The different...

FISH for Aneuploidy Screening

Cytogenetic studies have shown that up to 60 of the early spontaneous abortions in humans are caused by numerical chromosomal abnormalities and that as many as 70 of all human preimplantation embryos may contain aneuploid cells. 5 This prompted the development of PGD for aneuploidy screening of oocytes and preimplantation embryos PGD-AS . Fluorescent in situ hybridization is Fig. 2 Polar body biopsy. Removal of the first and second polar body. Fig. 2 Polar body biopsy. Removal of the first and...

Assay Formats Direct Hybridization

Oligonucleotide Ligation Assay Principle

Several formats of wet-ware were adopted to bead-based nucleic acid testing. The simplest method is the direct hybridization of labeled nucleic acids to beads carrying a sequence-specific oligonucleotide. An assay for gene expression analysis using color-coded microspheres was developed using this technique. In Ref. 4 , labeled cRNA was generated which was hybridized to a set of 20 bead populations carrying specific capture sequences of 25 bp for 20 Arabidopsis genes. The obtained expression...

Molecular Beacons

Stem Loop Pcr

Molecular beacon MB is a stem-loop DNA oligonucle-otide, which carries a fluorophore and a quencher at the 3'- and 5'-ends 4,5 Fig. 1A . In the absence of the target, these molecules form closed stem-loop structures in which fluorophore and quencher are in close proximity this results in fluorescence quenching. In the presence of the DNA or RNA target, molecular beacon forms a complex with it, which brings apart the fluorophore and the quencher. Once the fluorophore and quencher are spatially...

Molecular Diversity Of

Von Willebrand disease is an extremely heterogeneous disorder, and results from phenotypic and genotypic tests form the basis of the diagnosis and classification of different types of VWD. The numerous variants of VWD generally fit in the original classification of either type I partial and III total reduction or type II with qualitative defect of VWF. Multimeric analysis of von Willebrand antigen VWF Ag by SDS-gel agarose gel electrophore-sis 7 results in identification of many different type...

Optimization Of Liposomal Delivery

Mlv Liposome

Efficient in vivo nucleic acid-liposome complexes have unique features, including their morphology, mechanisms for cell and nuclear entry, targeted delivery, and ability to penetrate across tight barriers and throughout target tissues. Liposomes have different morphologies based on their composition and the formulation method that contribute to their ability to deliver nucleic acids in vivo. Formulations frequently used for the delivery of nucleic acids are lamellar structures, including small...

Distance Dependence Of Fret

Distance Dependence Fret

Fluorescence resonance energy transfer efficiency is inversely proportional to the sixth power of the distance between a donor and acceptor, and therefore is extremely Fig. 1 A General mechanisms of excited-state deactivation of a fluorophore. Up arrow represents transition from ground state S0 to an Si excited state thick line or higher thin lines after photon absorption wavy upward arrow . Down arrows represent relaxation, first from higher states down to Si short arrow . Relaxation from S1...

Microscopic Examination

In the past, surveys for lymphatic filariasis depended on the examination of blood films, which, in most areas, had to be collected around midnight. The thick blood smear technique normally employed to detect microfilaria is intensive especially when the parasite is nocturnally periodic and a nocturnal specimen is required to increase the chance of detecting the infection. The need for a nocturnal blood sample has been obviated by the administration of 50 and 100 mg of DEC before blood...

Dna Rearrangements Detected By D4f104s1 And D4z4 Repeats

P13e11 Probe

When probe p13E-11 D4F104S1 is hybridized against genomic DNA digested with the restriction enzyme EcoRI, it identifies a 3.3-kb tandem repeat sequence termed D4Z4 Fig. 1 . 4 The variability in the EcoRI fragment size is because of the deletion of an integral number of D4Z4 repeats. The D4F104S1 EcoRI polymorphism is complex and represents contributions from two separate homologous EcoRI polymorphic loci one located at 4q35, and the other at 10q26. Both polymorphic elements are composed of a...

Immunological Diagnosis

Intradermal tests performed by several investigators have produced equivocal results. Early studies with purified antigenic extracts of adult Dirofilaria immitis Sawada antigen showed high positive rates in W. bancrofti microfilaremic and clinically positive patients. 5 However, it was found that this antigen could not discriminate between patients with and without clinical manifestations. In addition, the Sawada antigen was reported to have cross-reactivity with other filarial infection. 6...