Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs.

PURPOSE: The primary method for analysis of low template DNA (LTDNA) is known as the low copy number (LCN) method involving an increased number of PCR cycles (typically 34). In common with other LTDNA methods, LCN profiles are characterized by allelic imbalance, drop in, and drop out that require complicated interpretation rules. They often require replicate PCR reactions to generate a "consensus" profile in a specialized facility. An ideal method for analysis of LTDNA should enhance profiling outcomes without elevated error rates and be performed using standard facilities, with minimum additional cost. METHODS: In this study, we present a comparison of four method variations for the amplification of STRs from LTDNA with a widely used, commercially available kit (AmpFℓSTR(®) Profiler Plus(®)): the standard method, the standard method with a post-PCR clean up, the LCN method, and a reduced reaction volume with increased Taq DNA polymerase concentration. RESULTS: Using telogen hairs-a common source of LTDNA-and matched reference DNA, the LCN method produced the highest number of concordant and non-concordant (i.e., dropped-in) alleles. In comparison, the reduced reaction volume with increased Taq polymerase yielded more full and concordant DNA profiles (all alleles combined) and less off-ladder alleles from a broad range of input DNA. In addition, this method resulted in less non-concordant alleles than LCN and no more than for standard PCR, which suggests that it may be preferred over increased PCR cycles for LTDNA analysis, either with or without consensus profiling and statistical modelling. CONCLUSIONS: Overall, this study highlights the importance and benefit of optimizing PCR conditions and developing improved laboratory methods to amplify and analyze LTDNA.

Pinot blanc and Pinot gris arose as independent somatic mutations of Pinot noir.

Somatic mutation is a natural mechanism which allows plant growers to develop new cultivars. As a source of variation within a uniform genetic background, it also represents an ideal tool for studying the genetic make-up of important traits and for establishing gene functions. Layer-specific molecular characterization of the Pinot family of grape cultivars was conducted to provide an evolutionary explanation for the somatic mutations that have affected the locus of berry colour. Through the study of the structural dynamics along chromosome 2, a very large deletion present in a single Pinot gris cell layer was identified and characterized. This mutation reveals that Pinot gris and Pinot blanc arose independently from the ancestral Pinot noir, suggesting a novel parallel evolutionary model. This proposed 'Pinot-model' represents a breakthrough towards the full understanding of the mechanisms behind the formation of white, grey, red, and pink grape cultivars, and eventually of their specific enological aptitude.

Exogenous contaminating DNA in Taq polymerases: A method to avoid false-positive results when detecting the blaTEM gene.

In the course of developing an assay to identify genes responsible for antibiotic resistance in gram-negative bacteria, it has been found that standard (not DNA-free) Taq DNA polymerases were contaminated with blaTEM gene fragments that varied in length and quantities. The complete blaTEM gene sequence was either absent or was detected in infinitesimal amounts. We developed an approach to avoid false-positive findings caused by contaminating blaTEM gene sequences in conventional polymerases. The method is based on selection of a target sequence to be detected within the blaTEM gene in such a way that the chosen sequence is amplified with primers incapable of amplifying contaminating DNA sequences of the polymerase.

Allelic loss analysis by denaturing high-performance liquid chromatography and electrospray ionization mass spectrometry.

Analysis of allelic imbalance is of great importance for understanding tumorigenesis and the clinical management of malignant disease. Fluorescent-based capillary electrophoresis (CE) of highly polymorphic short tandem repeats (STRs) has become the main method used to detect the loss/gain of alleles. However, there is continued interest in the development of techniques that require no fluorescence and allow the rapid analysis of individual samples. One promising alternative is ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), which is widely available because of its use in denaturing HPLC. Its applicability in combination with ultraviolet (UV) absorbance detection to the efficient separation of di- and tetranucleotide repeats on the short arm of chromosome 11 was tested using 25 matched pairs of normal and ovarian cancer tissues. Loss of heterozygosity (LOH) could be readily identified for all 13 loci tested, based on changes in the ratios between either the alleles or homo- and heteroduplex signals. However, discrimination between noninformative homo- or hemizygous and heterozygous samples was difficult or impossible when HPLC failed to resolve the alleles. Hyphenation of HPLC with electrospray ionization (ESI) quadrupole ion trap (IT) mass spectrometry (MS) not only allowed the identification of coeluting alleles, but also the reliable detection of a 40% reduction of one allele. The size range of DNA fragments amenable to mass spectrometric analysis was effectively tripled to >300 bp by the use of a linear IT and a Taq DNA polymerase cocktail lacking detergents that otherwise adversely affect ESI.

Detection of the C282Y and H63D polymorphisms associated with hereditary hemochromatosis using the ABI 7500 fast real time PCR platform.

Classic hereditary hemochromatosis is an autosomal recessive disorder characterized by iron overload and sequence variants in the HFE gene. The HFE gene is located at 6p21.3 and contains 2 common single nucleotide polymorphisms (SNPs) C282Y and H63D, which are routinely tested for in the molecular diagnostics laboratory. In this study, we used DNA samples from 59 patients in which clinicians wanted to confirm or rule-out hereditary hemochromatosis that had been previously tested for the HFE SNPs using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and the ABI 7700 real time PCR assay with a MGB Eclipse ASR Probe system. The new assay used TAQman SNP Genotyping Assays, which were performed on the ABI 7500 FAST real time PCR platform. Allelic discrimination was determined during a postamplification plate read. Of the 59 samples genotyped, 7 were homozygous for C282Y, 6 were heterozygous for C282Y, 9 were homozygous for H63D, 10 were heterozygous for H63D, 6 were compound heterozygotes, and 20 were wild type. With the exception of one sample that was indeterminate by the TAQman SNP Genotyping Assay, all others showed 100% concordance between the 3 assays. The one indeterminate sample was heterozygous for C282Y by the PCR-RFLP and ABI 7700 real time PCR assays, but there was an insufficient quantity of DNA to perform the TAQman SNP Genotyping Assay. Our study suggests that the ABI 7500 FAST TAQman SNP Genotyping Assay is comparable with the PCR-RFLP and ABI 7700 real time PCR methods in detecting and characterizing these 2 HFE SNPs. Improved software and thermocycling capabilities have resulted in a very robust TAQman assay with the advantage of a much improved turn-around-time and throughput.

Fluorescence-based, high-throughput DNA polymerase assay.

The commonly used DNA polymerase assay is based on the detection of incorporated radiolabeled nucleotides in a DNA elongation reaction. It is laborious, radioactive, and can be highly variable. Here we report a nonradioactive fluorescence-based assay. The method consists of Cydye-labeled nucleotides, biotinylated primer, and a streptavidin-coated microplate. The assay is found to have sensitivity and dynamic range comparable to the classical radioactive method. Moreover, it has the advantages of being simple, stable, nonradioactive, and suitable for high-throughput applications. We have also found that, to ensure efficient measurement of the enzyme activity, the template DNA used in this method should have a sequence that avoids the incorporation of the fluorescence-labeled nucleotide in a consecutive way.

Improving the display of proteins on filamentous phage.

In phage display technology, polypeptides are displayed on the surface of filamentous bacteriophage by genetic fusion to a coat protein. However, the fraction of phage particles bearing the fusion protein can be low. Here we found that we could improve the display of a protein (Stoffel fragment of Taq polymerase fused to the p3 protein of the phage) by mutation of the signal sequence and use of helper phage with a protease-cleavable coat protein. Over multiple rounds of infection, proteolysis and binding to an anti-Taq antibody, we were able to select strongly for display of the fusion protein (> 50-fold), and for mutations in the translation initiation region and in the signal sequence of the fusion. This suggests a general means of improving the display of proteins on phage.

Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children.

BACKGROUND: Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples. METHODS: A serum sample was available from patients classified serologically as primary EBV infection (n = 28), EBV-seronegative (n = 25) and EBV-seropositive (n = 26). Patients were classified as having EBV infectious mononucleosis if they had specified clinical findings and > or =10% atypical lymphocytes in peripheral blood or had a positive Monospot test result. DNA was purified by a spin column method and tested in PCR reactions with primers for EBV DNA polymerase gene and internal control targets. Amplification of the two PCR products was measured in real time with separate TaqMan DNA probes labeled with various fluorescent reporters. RESULTS: The mean age of study patients was 9 years, 4 months. Twenty-one (75%) of the patients in the primary EBV infection group, one (4%) of the seronegatives and none of the seropositives had detectable EBV DNA. Within the primary infection group, those with detectable virus were more likely than those without detectable virus to have evidence of lymphadenopathy (14 of 16 vs.1 of 5; P = 0.011), higher mean atypical (11.7 vs.0.9%; P = 0.002) and absolute atypical (1.5 vs.0.1 x 109/l; P = 0.004) lymphocyte count, higher mean absolute lymphocyte count (4.7 vs.2.3 x 109/l; P = 0.026) and higher mean aspartate aminotransferase value (119.8 vs.37.3 IU/l; P = 0.036). Ten patients, all in the primary infection group, had EBV infectious mononucleosis, and all had positive PCR results. No sample contained PCR inhibitors. CONCLUSIONS: A real time TaqMan PCR assay allows rapid identification of patients with primary EBV infection and those with EBV infectious mononucleosis.

[Prenatal diagnostics of Toxoplasma gondii invasion by quantitative method of PCR-TaqMann]. / Prenatalna diagnostyka zarazenia pierwotniakiem Toxoplasma gondii metoda ilosciowej polimerazowej reakcji lancuchowej PCR-TaqMann.

Routine serological diagnosis of toxoplasmosis provides high sensitivity, but not high specificity. The high sensivity combined with high specifity offered by PCR-TaqMann as well as the degree of infection led us to investigate the presence and levels of T. gondii DNA in amniotic fluid, maternal and neonatal blood in cases of pregnancy where infection with this agent was suspected. Samples of amniotic fluid and blood were taken from pregnant women. Postnatal blood samples were also taken from their infants. Presence and levels of toxoplasma DNA was investigated using PCR-TaqMann. PCR products were detected by electrophoresis on polyacrylamide gel. The PCR-TaqMann test is highly sensitive, specific and useful method allowing detection of the parasite genome and assessement of its level.

Comparison between a TaqMan polymerase chain reaction assay and a culture method for ctx-positive Vibrio cholerae detection.

The main objective of the present work was to evaluate a real-time polymerase chain reaction (PCR) method to detect toxigenic Vibrio cholerae in Pangasius hypophthalmus, a freshwater fish cultured mainly in South East Asia. A FDA traditional culture method and a real-time PCR method of the ctx gene were used for detection of V. cholerae in spiked samples of pangasius fish. After an overnight enrichment of samples at 37 degrees C in alkaline peptone water, 2 cfu/25 g of fish was detected with both methods. Although both methods were very sensitive, obtaining results with culture methods may take several days, while real-time PCR takes only a few hours. Furthermore, with traditional methods, complementary techniques such as serotyping, although not available for all serogroups, are needed to identify toxigenic V. cholerae. However, with real-time PCR, toxigenic serogroups are detected in only one step after overnight enrichment.

Performance evaluation of five commercial real-time PCR reagent systems using TaqMan assays for B. anthracis detection.

BACKGROUND: Real-time PCR assay sensitivity is affected by the choice and concentrations of reaction mix constituents among other factors such as primers, probes, and analytical assay platforms. Commercially available reagent mixes facilitate PCR assay set-up with fewer steps and timeliness. However, determination of analytical assay framework is important for ready-to-use real-time PCR reagent systems for rapid, quantitative and accurate detection of bioterror pathogens such as Bacillus anthracis. METHODS: In this study, performance characteristics of five commercially available quantitative PCR reagent mixes were evaluated using TaqMan-based real-time PCR. The reagent systems were tested for compatibility on the ABI 7000 assay platform and compared for their distinctive analytical characteristics using the B. anthracis rpoB and pag gene real-time PCR assays. RESULTS AND CONCLUSIONS: Knowledge of distinctive assay performance characteristics of commercially available qPCR reagent mixes is critical for carefully designing analytical assay systems. The ABI, ABgene and Eppendorf reagent systems performed consistently overall for the two TaqMan assays for B. anthracis detection that were used in the current study. However, the use of Eppendorf reagent system requires shorter thermal cycling time. In addition, while the ABI and Eppendorf systems have similar assay sensitivity for both the rpoB and pag assays, the Eppendorf system achieves the same with lower C(T) values.

Comprehensive screening of multiple epiphyseal dysplasia mutations in Japanese population.

Multiple epiphyseal dysplasia (MED) is among the most genetically heterogeneous skeletal dysplasias. Six genes involved in MED, COMP, MATN3, COL9A1, COL9A2, COL9A3, and DTDST have been identified; however, the presence of additional disease genes has been reported, and the detection rate for mutations in known genes accounts for no more than 50% of patients with MED in Western populations. Here, we screened the six known disease genes in 35 consecutive Japanese MED patients. We analyzed the entire coding region of each gene, along with flanking intron-exon junctions, by direct sequencing. A total of 19 mutations were identified in COMP, MATN3, COL9A2, COL9A3, and DTDST. The detection rate for known mutations was higher in this study than in previous reports, and we identified a substantially different spectrum of mutations. Mutations in MATN3 were more prevalent among these Japanese patients, whereas no DTDST mutations were detected. Most of the mutations were localized within specific regions of each gene: COMP mutations were found in the calmodulin-like repeat domains; MATN3 mutations in the von Willebrand factor type A domain; and type IX collagen gene mutations occurred in the third collagenous domains. Based on the integration of clinical and genetic information, we propose an algorithm for detecting mutations in Japanese MED patients. Our study further supports the existence of additional MED gene(s).

MAPKAP kinase 2-deficient mice are resistant to collagen-induced arthritis.

TNF-alpha is a pleiotropic cytokine considered a primary mediator of immune regulation and inflammatory response and has been shown to play a central role in rheumatoid arthritis (RA). MAPKAP kinase 2 (MK2) is a serine/threonine kinase that is regulated through direct phosphorylation by p38 MAPK, and has been shown to be an essential component in the inflammatory response that regulates the biosynthesis of TNF-alpha at a posttranscriptional level. The murine model of collagen-induced arthritis (CIA) is an established disease model to study pathogenic mechanisms relevant to RA. In this study, we report that deletion of the MK2 gene in DBA/1LacJ mice confers protection against CIA. Interestingly, the MK2 heterozygous mutants display an intermediate level of protection when compared with homozygous mutant and wild-type littermates. We show that MK2(-/-) and MK2(+/-) mice exhibit decreased disease incidence and severity in the CIA disease model and reduced TNF-alpha and IL-6 serum levels following LPS/d-Gal treatment compared with wild-type mice. Additionally, we show that levels of IL-6 mRNA in paws of mice with CIA correlate with the disease status. These findings suggest that an MK2 inhibitor could be of great therapeutic value to treat inflammatory diseases like RA.

Fluorescence resonance energy transfer-based detection of analytes using antiidiotypic affinity protein pairs.

A new method for specific detection of proteins based on fluorescence resonance energy transfer (FRET) using affinity proteins (affibodies) derived from combinatorial engineering of Staphylococcal protein A has been developed. Antiidiotypic affibody pairs were used in a homogeneous competitive binding assay, where the idiotypic, target-specific affibody was labeled with fluorescein and the antiidiotypic affibody was labeled with tetramethylrhodamine. Intermolecular FRET between the two fluorescent probes was observed in the antiidiotypic affibody complex, but upon addition of target protein the antiidiotypic affibody was displaced, which was monitored by a shift in the relative emission of the donor and acceptor fluorophores. The feasibility of the system was demonstrated by the detection of IgA and Taq DNA polymerase with high specificity, using two different antiidiotypic affibody pairs. Detection of Taq DNA polymerase in 25% human plasma was successfully carried out, demonstrating that the method can be used for analysis of proteins in samples of complex composition.

Escherichia coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ.

Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.

The role of cyclooxygenase 2 in ulcerative colitis-associated neoplasia.

Cyclooxygenase 2 (COX-2) overexpression has been described in sporadic colonic neoplasia, but its role in ulcerative colitis (UC) neoplastic progression remains unexplored. Although the specific role of cyclooxygenase in colonic neoplasia is uncertain, its inhibition by nonsteroidal anti-inflammatory drugs decreases the risk of sporadic colonic adenocarcinoma and causes regression of adenomas in familial adenomatous polyposis. To investigate the role of COX-2 in UC-associated neoplasia, we assessed COX-2 protein and mRNA expression throughout the spectrum of UC-associated neoplastic lesions in four total colectomy specimens, using immunocytochemistry and a novel TaqMan reverse transcriptase-polymerase chain reaction assay. The findings were correlated with DNA ploidy and inflammatory activity. We found COX-2 overexpression throughout the neoplastic spectrum in UC (P: < 0.0001, R:(2)=0.53), even in diploid samples that were negative for dysplasia. Overall, neoplastic change explained 53% of the variation in COX-2 expression, whereas inflammatory activity explained only 11%. COX-2 was overexpressed in all aneuploid samples and in 38% of diploid samples (P: = 0.0074). cDNA representational difference analysis was also performed and revealed that COX-2 mRNA was an up-regulated cDNA representational difference analysis difference product. COX-2 overexpression occurs early in UC-associated neoplasia, and the increase cannot be explained by inflammatory activity alone. The data suggest that COX-2-specific inhibitors may have a chemopreventative role in UC but the possibility that they could exacerbate UC inflammatory activity needs to be tested.

Elimination of bacterial DNA from Taq DNA polymerases by restriction endonuclease digestion.

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 fg of bacterial DNA. This method can be readily adapted to suit other sensitive PCRs required for clinical applications.