Thermal Cycling Parameters Research Articles

Microbial DNA Typing by Automated Repetitive-Sequence-Based PCR

Repetitive sequence-based PCR (rep-PCR) has been recognized as an effective method for bacterial strain typing. Recently, rep-PCR has been commercially adapted to an automated format known as the DiversiLab system to provide a reliable PCR-based typing system for clinical laboratories. We describe the adaptations made to automate rep-PCR and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. The modifications for automation included changes in rep-PCR chemistry and thermal cycling parameters, incorporation of microfluidics-based DNA amplicon fractionation and detection, and Internet-based computer-assisted analysis, reporting, and data storage. The performance and reproducibility of the automated rep-PCR were examined by performing DNA typing and replicate testing with multiple laboratories, personnel, instruments, DNA template concentrations, and culture conditions prior to DNA isolation. Finally, we demonstrated the use of automated rep-PCR for clinical laboratory applications by using isolates from an outbreak of Neisseria meningitidis infections. N. meningitidis outbreak-related strains were distinguished from other isolates. The DiversiLab system is a highly integrated, convenient, and rapid testing platform that may allow clinical laboratories to realize the potential of microbial DNA typing.

Constructing Universal Multiplex Pcr Systems for Comparative Genotyping

Analysis of length polymorphisms at STR loci in the human genome has become a standard approach for comparative genotyping in many areas including disease research and diagnostics, parentage assessment, investigations of human diversity, and forensic science. The simultaneous analysis of multiple STR loci through multiplex PCR and multicolor fluorescence detection offers sample conservation, high throughput, and automated genetic analysis. Careful design and optimization of tetranucleotide STR multiplexes has led to reliable, standardized systems that powerfully differentiate and distinguish individual human DNA profiles. The development of these multiplex systems involved a rigorous experimental strategy that included careful selection of PCR primer sequences (for yield, specificity, and multiplex compatability), along with optimization of PCR component concentrations, thermal cycling parameters, and fluorescence detection conditions. This developmental approach rendered well-characterized DNA typing systems that are high performing (sensitive, specific, and balanced), optimized to universal parameters (same reaction conditions), resilient to fluctuations in reaction conditions, and simple to implement and use routinely.

A generic nested-RT-PCR followed by sequencing for detection and identification of members of the alphavirus genus

A specific and sensitive nested RT-PCR method was developed for the detection of members of the alphavirus genus. Based on available sequences, degenerated primers were selected in the nsP4 gene. Reaction components and thermal cycling parameters were investigated and standardised, and optimal ones were selected. As few as 25 pfu/tube could be detected. The identities of the amplified fragments were confirmed by sequencing, and phylogenetic analysis was carried out. The resulting phylogenetic tree could be applied to classify every alphavirus according to its serogroup. This technique is suitable for rapid, sensitive and reliable detection of these viruses and may be very valuable for diagnostic applications and surveillance.

Detection and typing of lymphotropic herpesviruses by multiplex polymerase chain reaction

A multiplex nested-polymerase chain reaction (PCR) method was developed for the simultaneous detection and typing of all human lymphotropic herpesviruses described to date, including Ebstein–Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6, variants A and B (HHV6-A, HHV6-B), human herpesvirus 7 (HHV7) and human herpesvirus 8 (HHV8). Oligonucleotide primers were designed to amplify a highly conserved region within the DNA polymerase gene. Each reaction component and thermal cycling parameters were thoroughly standardized to achieve optimal specificity and sensitivity for the PCR assay, which was estimated at about 10–100 molecules for each virus. An internal control, consisting of 100 molecules of a cloned fragment of the porcine pseudorabies herpesvirus (PrV) genome, was included to detect false negative results. To assess suitability and clinical application of the multiplex PCR method, a total of 35 well-characterized specimens, including Kaposi’s sarcoma skin lesions, serum, cerebrospinal fluid, saliva and urine samples, were tested. Results obtained suggest this technique could be applied as a sole diagnostic tool in several clinical settings in which herpesviral infection is suspected and differential diagnosis required, avoiding the need to test specimens by separate PCR methods.

DAF optimization using Taguchi methods and the effect of thermal cycling parameters on DNA amplification.

Taguchi methods, which are widely applied in industrial process design, were used to optimize DNA amplification finger-printing (DAF). Quadratic loss functions that penalize deviations from prediction values and L9 (3(4)) and L18 (3(8)) orthogonal arrays revealed effects and interactions of amplification reaction components and thermal cycling parameters. Analysis of variance (ANOVA) decomposed the contribution of individual factors to the experimental response (amplification yield and product number), while verification experiments established that optimum conditions were predictable, verifiable and reproducible. While several amplification components (primer, magnesium and enzyme) conditioned the amplification reaction, annealing temperature and time were the only important thermal cycling contributing factors. The Taguchi strategy defined a robust and transportable amplification protocol based on high annealing temperatures (typically 48 degrees C) and primer concentrations (typically 8 microM), which can be applied to the fingerprinting of a wide range of DNA templates of plant and fungal origin. The general strategy of robust experimental design holds potential as an optimization tool for other methods in molecular biology.

TWGDAM Validation of the AmpFISTR™ Blue PCR Amplification Kit for Forensic Casework Analysis

Studies were performed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) committee to validate the AmpFISTR Blue PCR Amplification Kit for forensic casework applications. The kit coamplifies the tetranucleotide short tandem repeat (STR) loci D3S1358, vWA, and FGA. The dye-labeled amplification products were electrophoresed and detected directly using the ABI PRISM 377 DNA Sequencer or the 310 Genetic Analyzer. CEPH family studies demonstrated Mendelian inheritance of these loci and probability of identity values from population studies were 1/4,830 (African-American), 1/5,479 (U.S. Caucasian), and 1/3,443 (U.S. West Coast Hispanic). In all studies examining different body tissues and fluids, the expected genotypes were observed. Studies to determine and test the PCR reagent components and thermal cycling parameters demonstrated specificity, sensitivity, and balance over a wide range of conditions. Reliable results were obtained from DNA quantities as low as 0.25 ng. A variety of environmental studies were performed, as forensic samples are often exposed to different environmental conditions and substances which may degrade DNA or inhibit the amplification process. Highly degraded samples demonstrated that FGA was the first locus to become undetectable, followed by vWA, and then D3S1358; this is the expected pattern according to locus size. In studies of PCR inhibition, the pattern in which the loci became undetectable was different; FGA was the first locus to become undetectable, followed by D3S1358, and then vWA. Single versus multiple locus amplifications revealed no benefit to single locus analysis, even in cases of degradation or inhibition. The occurrence of preferential amplification was very rare, particularly in noncompromised, unmixed samples. Artifact peaks were not observed in any instance. Mixture studies confirmed the ability to detect mixed DNA samples and included the characterization of stutter and peak height ratios; the limit of detection was 1:10 for 1 ng total genomic DNA and 1:30 for 5 ng. DNA extracted from nonprobative case evidence was successfully amplified and genotyped. All such studies indicate that the AmpFISTR Blue PCR Amplification Kit will reproducibly yield specific and sensitive results.

Selective amplification of RNA utilizing the nucleotide analog dITP and Thermus thermophilus DNA polymerase.

The ability to selectively amplify RNA in the presence of genomic DNA of analogous sequence is cumbersome and requires implementation of critical controls for genes lacking introns. The convenient approaches of either designing oligonucleotide primers at the splice junction or differentiating the target sequence based on the size difference obtained by the presence of the intron are not possible. Our strategy for the selective amplification of RNA targets is based on the enzymology of a single thermostable DNA polymerase and the ability to modulate the strand separation temperature requirements for PCR amplification. Following reverse transcription of the RNA by recombinant Thermus thermophilus DNA polymerase (rTth pol), the resulting RNAxDNA hybrid is digested by the RNase H activity of rTth pol, allowing the PCR primer to hybridize and initiate second-strand cDNA synthesis. Substitution of one or more conventional nucleotides with nucleotide analogs that decrease base stacking interactions and/or hydrogen bonding (e.g. hydroxymethyldUTP or dITP) during the first- and second-strand cDNA synthesis step reduces the strand separation temperature of the resultant DNAxDNA duplex. Alteration of the thermal cycling parameters of the subsequent PCR amplification, such that the strand separation temperature is below that required for denaturation of genomic duplex DNA composed of standard nucleotides, prevents the genomic DNA from being denatured and therefore amplified.

The high temperature behaviour of long fibre reinforced titanium under transverse loading

Isothermal and thermal cycling creep tests have been performed on titanium matrix composites reinforced with continuous SiC fibres under transverse loading. Isothermal results have shown that the creep exponent of the reinforced matrix is much lower than that of the unreinforced matrix. Influence of thermal cycling parameters is examined and comparison is made with the results of isothermal tests. It is demonstrated that stress relaxation in the matrix and interfacial damage play a key role. It is also shown that post-fabrication heat treatment greatly improves the high temperature mechanical properties of the composites. After a detailed analysis of the interface, the different mechanisms of damage have been identified. Evidence is given that the properties of the carbon and TiB 2 layers, originally present at the fibre surface, are responsible for the low resistance to damage of the as-fabricated materials under transverse loading.

Characterization of human AFLP systems apolipoprotein B, phenylalanine hydroxylase, and D1S80.

Methodology is presented for amplified fragment length polymorphism (AFLP) typing using a nonisotopic, PCR protocol. Human variable number tandem repeat (VNTR) loci used for identification in forensic and paternity testing were optimized for reaction and thermal-cycling parameters. Loci analyzed were the apolipoprotein B (APOB) 3' hypervariable region (HVR), phenylalanine hydroxylase 3' HVR (PAH), and D1S80. Coamplification of a monomorphic beta-globin fragment serves as an amplification control. Biotin is integrated into PCR amplicon through primer incorporation. AFLP products undergo agarose gel electrophoresis and Southern transfer to a nylon membrane. Amplicons were detected using a streptavidin-enzyme conjugate. Either colorimetric- or chemiluminescent-developed bands are genotyped using locus-specific allele ladders with known VNTR repeat numbers. Using this methodology, we have successfully typed > 500 individuals from three population groups for each locus during data basing and casework.

A guide to the design and use of mismatched and degenerate primers.

Roche Molecular Systems, Inc., Alameda, California 94501 Although simple in concept, PCR requires a myriad of complex interactions between template, primers, deoxynucleoside triphosphates, and DNA polymerase to successfully accomplish targeted amplification. Primer design in conjunct ion with changes in the concentrat ions of reaction components and thermal cycling parameters provides a versatility available in no other molecular technique. For example, PCR can be employed to accommodate mismatches in the pr imertemplate duplex, thereby permit t ing amplification not only of related sequences but also of uncharacterized sequences. It can also be designed to amplify selectively a small number of mutan t genes with single base alterations in a vast background of normal genes. The flexibiliW of PCR has greatly simplified molecular manipulat ions. Procedures for altering a particular template sequence, which in the past required several steps, can now be performed with only a few manipulat ions. Because 3' and internal mismatches between pr imertemplate duplexes are tolerated under appropriate conditions, point mutat ions and desired restriction endonuclease sites can be directly introduced into the primers. Likewise, nucleotide insertions and deletions can be similarly introduced into the amplified product via the primers. Degenerate primers have made it possible to amplify related but distinct nucleic acid sequences as well as to amplify targets for which only amino acid sequences are available. The range of possible applications precludes providing detailed protocols for each. Instead, the delineation of the critical parameters and a guide for representative use will be summarized. Depending on the application, a subset of these recommendat ions or suggestions may suffice.

Microstructural developments in Mg-Ti-PSZ systems

The microstructure of titania-added Mg-partially-stabilized zirconia (PSZ) is dramatically influenced by thermal treatments. Effects of various sintering, heat-treatment and thermal shock cycling parameters on the microstructure of the Mg-Ti-PSZ system are described. Conditions favourable for the growth of needle-like Ti-rich reinforcements in highly thermalshock-resistant Mg-Ti-PSZ ceramics are identified. TiO2 seems to play a catalytic role in the formation of Zr-rich networks during high-temperature (1700°C) sintering of the Mg-Ti-PSZ system, quite similar to those found in Mg-PSZ, heat-treated above 1300 °C.