Template Mixtures Research Articles

Optimization of a duplex amplification and sequencing strategy for the HVI/HVII regions of human mitochondrial DNA for forensic casework

A duplex primer set for the amplification of mitochondrial DNA HVI and HVII control regions was evaluated for the optimization of a DNA sequencing protocol suitable for forensic casework. HVI and HVII products, with the absence of non-specific products, could be detected by agarose gel electrophoresis when as little as 0.5 and 0.1 pg of DNA were amplified for 34 and 38 cycles, respectively. Because HVI and HVII amplicons are co-synthesized in the duplex PCR, fewer steps are required (lessening the risk of cross contamination events) and more frugal use of precious extracted DNA samples is possible, both desirable features for forensic casework. The ABI Prism ® BigDye™ version 1.1 chemistry provided high quality sequencing data, with little or no background noise and uniform peak heights, outcomes that favored reliable detection of heteroplasmy, particularly at early sequence reads (<40 bases). Optimal compromise between sensitivity and sequence accuracy in the absence of noise was achieved starting at 150 mitochondrial genome copies. The protocol is effective (no sequence errors) with highly degraded DNA (average detectable template size of 200 bp). Dual artificial template mixtures with the minor component at 15% suggests that heteroplasmy should be detected at this level with confidence.

Optical Properties of Nanostructured Mesoporous Semiconductor Films

AbstractDirect liquid crystal templating from non-ionic polyoxyethylene surfactants has been utilised to produce well-defined birefringent films of nanostructured cadmium telluride with mesoporous architectures of extended spatial periodicities. The template mixtures and films were characterised by X-ray diffraction, transmission electron microscopy and polarising optical microscopy to ascertain the presence of a regular nanostructure. UV-VIS reflectance spectroscopy was employed to investigate the films' optical properties.

Adsorption isotherms of a molecular imprinted polymer prepared in the presence of a polymerisable template: Indirect evidence of the formation of template clusters in the binding site

The current opinion about molecular imprinted polymers (MIPs) is that their molecular recognition properties are due to the presence of nanocavities formed during a polymerization process developed in the presence of a template molecule. According to this principle, the shape of these nanocavities is complementary to that of the template and non-covalent interactions are established between the binding site and a single template molecule. Nevertheless, there are some experimental indications that the real molecular recognition mechanism involves clusters of template molecules being packed into the binding site. Recently, it has been proposed that template molecules covalently linked to the binding site can act as nucleation points, enhancing the formation of these molecular clusters. We have tested this hypothesis by studying the adsorption isotherms of polymers prepared by imprinting them with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Three different polymers were considered: P0, prepared without the template, P1, whose template was represented by 2,4,5-T molecules, and P2, whose template was 1/3 constituted by the polymerisable 2-(2,4,5-trichlorophenoxyacetoxy)-ethylmethacrylate (2,4,5-TEMA) and 2/3 by 2,4,5-T. The polymers were prepared by thermoinduced polymerization of template mixtures, 4-vinylpyridine and ethylene dimethacrylate. The crushed polymers were packed into HPLC columns and frontal chromatographic runs were performed by eluting the columns with a mobile phase containing variable amounts of 2,4,5-T. The experimental adsorption isotherms were fitted by using several isotherm models, and the Freundlich–Langmuir model was found to give the best fitting in terms of F-test. All the models considered showed a significant difference between the affinity constant values measured for the polymer P1 and P2, with a higher value for the polymer P2 (for Freundlich–Langmuir model: polymer P1, k=(2.00±0.43)×10 4 M −1; polymer P2, k=(1.93±0.0535)×10 5 M −1; ratio P2/P1, 9.65±2.09). Such experimental results support the hypothesis that a polymer prepared with a limited amount of template covalently attached to the binding site shows an increased affinity for the template itself.

Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.

Mutagenically separated PCR assay for rapid detection of M41L and K70R zidovudine resistance mutations in CRF01_AE (subtype E) human immunodeficiency virus type 1.

A rapid zidovudine (ZDV) resistance genotypic assay was developed based on the mutagenically separated PCR (MS-PCR) technique to detect two ZDV-resistant mutations, M41L and K70R in CRF01_AE (subtype E). Endpoint dilution analysis revealed that the newly constructed MS-PCR assay could successfully detect three to nine copies of human immunodeficiency virus type 1 template RNA. The test against wild-type and mutant template mixtures in different ratios demonstrated that the assay could detect 10% minor population, at least. Fifty-one subtype E clinical samples were analyzed by the newly constructed MS-PCR assay and direct nucleotide sequencing. The concordance of the two assays was 92 and 100% in codons 41 and 70, respectively. The MS-PCR assay is a rapid, simple, and inexpensive assay that is highly sensitive in detecting mutant targets, including minor populations. Thus, it could be used as a powerful tool for epidemiological surveillance of drug-resistant mutations in developing countries.

Electrodeposition and properties of nanostructured platinum films studied by quartz crystal impedance measurements at 10 MHz

The electrodeposition of platinum films from aqueous solutions and lyotropic liquid crystalline mixtures of the non-ionic surfactant octaethyleneglycol monohexadecyl ether, water, and hexachloroplatinic acid (HCPA) has been studied by rapid in-situ measurements of the quartz crystal electroacoustic impedance with an electrochemical quartz crystal microbalance (EQCM). For solutions and liquid crystalline template mixtures of high HCPA content large changes of the damping resistance, R have been observed at the initial stages and the end of the plating process as a result of composition changes in the depletion layer. They were corrected for to yield accurate in-situ mass data for the electroplating process. For the measured film thicknesses (<200 nm) the faradayic efficiency depends inversely on the concentration of HCPA in the aqueous solutions and the template mixtures. This was attributed mainly to migrational transport of the intermediate [PtCl 4] 2− anion away from the electrode and to the concentration dependence of the disproportionation/comproportionation reaction in the depletion layer between the Pt(0), Pt(II), and Pt(IV) species involved in the electrode reaction. A 65% increase in specific surface area was measured for the template deposited material compared to Pt deposits from aqueous solutions. This difference can be regarded as a minimum value for the thin films studied and becomes much larger for thicker films. Transmission electron microscopy (TEM) analysis of the former show the films to be mesoporous with a hexagonal nanostructure identical to that produced by template depositions onto non-oscillating substrates. The high specific surface area of the Pt films from lyotropic liquid crystalline templates results in a sensitivity enhancement of the in-situ electroacoustic impedance EQCM technique. It could hence be applied to mono- and sub-monolayer adsorption studies for polycrystalline Pt in sulfuric acid. An increase in damping resistance was found when stepping the potential from the double layer region into the hydrogen adsorption region and attributed to a higher degree of acoustic shear wave coupling to the bulk liquid. It is believed that the adsorbed hydrogen forms hydronium species, which others have observed earlier by IR spectroscopy.

Optimization of AFLP fingerprinting of organisms with a large-sized genome: a study on Alstroemeria spp.

The recently introduced PCR-based DNA fingerprinting technique AFLP (amplified fragment length polymorphism) allows the selective amplification of subsets of genomic restriction fragments. AFLP has been used for multiple purposes such as the construction of linkage maps, marker saturation at specific genomic regions, analysis of genetic diversity and molecular phylogeny and cultivar identification. AFLP can be tailored by varying the number of selective nucleotides added to core primers and can allow accurate amplification, even in complex template mixtures generated from plant species with very large genomes. In this study Alstroemeria, a plant species with a very large genome, was tested for adapting the AFLP protocol. The results indicated that the estimated number of amplification products was close to the observed number when eight selective nucleotides were used but that seven selective nucleotides did not increase the number of amplification products fourfold. However, we found reproducibility in both +7 and +8 fingerprints. Various distributions of selective nucleotides over the various rounds of preamplifications were tested. Preamplification with four selective nucleotides followed by final amplification with eight selective nucleotides produced clear and reproducible AFLP patterns. The effects of GC content of primers and multiple preamplification steps were also discussed.

PCR amplification of murine immunoglobulin germline V genes: strategies for minimization of recombination artefacts.

Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the 'plateau phase' of the reaction was reached. Recombination events were located throughout the approximately 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.

Fine Control of Pore Size of Highly Ordered MCM-41 by Using Template Mixtures of Dodecyltrimethylammonium Bromide/Hexadecyltrimethylammonium Bromide with Various Molar Ratios

Abstract MCM-41 materials were prepared hydrothermally by using template mixtures of hexadecyltrimethylammonium/dodecyltrimethylammonium (C16TMABr/C12TMABr) with various molar ratios. The MCM-41 materials thus prepared were highly ordered and their pore sizes can be finely controlled by changing the C16TMABr/C12TMABr molar ratio.

Quantitative Restriction Fragment Length Polymorphism: A Procedure for Quantitation of Diphtheria Toxin Gene CRM197 Allele

Here we present an assay for quantitation of a particular gene allele in DNA mixtures by means of restriction fragment length polymorphism (RFLP) in combination with polymerase chain reaction (PCR). We applied the quantitative RFLP principle for estimation of the relative amount of diphtheria toxin gene CRM197 allele inCorynebacterium diphtheriaeculture DNA samples. The procedure is based on PCR-mediated generation of an artificialAluI restriction site specifically with the CRM197 DNA template. AfterAluI digestion of the PCR product and polyacrylamide gel electrophoresis of the restriction fragments, the percentage of CRM197 template in the initial DNA sample was determined by scanning a gel negative. The method was shown to give a linear response when applied to template mixtures containing different amounts of CRM197 reference template. For samples where non-CRM197 DNA was detected byAluI RFLP, we designed a further allele-specific PCR assay to determine whether the non-CRM197 template portion was the wild-type toxin gene allele.

Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA

The MutEx assay is a technique that was developed to detect and map mutations. This assay takes advantage of the Escherichia coli mismatch binding protein MutS, which binds and protects mismatched, heteroduplex DNA from subsequent exonuclease digestion. The plausibility of using the MutEx assay as part of a genotypic selection scheme was investigated. Heteroduplexes were formed between mouse H- ras gene PCR products or restriction fragments that contained wild-type sequence and sequence with a single base change at codon 61 (wild-type, CAA and mutant, AAA). The heteroduplexes were incubated with MutS and then treated with the exonuclease activity of T7 DNA polymerase. MutS-protected DNA sequences were amplified by PCR. When this method was linked to single nucleotide primer extension (SNuPE) for mutant base identification, original mutant fractions of 1 in 50 000 and above were detected. Using comparable DNA template mixtures, the sensitivity of SNuPE alone was 1 in 5 or 1 in 50, depending on the direction of SNuPE priming and the particular base being incorporated. We conclude that the MutEx assay was able to enrich the mutant sequence approximately 1000-fold and, therefore, has considerable potential as a tool for mutation detection.