Evaluation of different machines used to quantify genetic modification by real-time PCR

Quantification of genetic modification (GM) is often undertaken to test for compliance with the European Union GM labeling threshold in food. Different control laboratories will often use common validated methods, but with different models of real-time PCR machines. We performed two separate ring trials to evaluate the relative precision and accuracy of different types of real-time PCR machines used to quantify the concentration of GM maize. Both trials used dual-labeled fluorogenic probes for quantification. The first ring trial used separate GM and reference assays (a single fluorescence channel), and the second used a combined duplex assay (two simultaneous fluorescence channels). Five manufacturers and seven models--including a 96-well microtiter-plate, rotary, and portable machines--were examined. In one trial, the machine used had a significant effect on precision, but in the other it did not. Overall, the degree of variation due to the machine model was lower than other factors. No significant repeatable difference in accuracy was observed between machine models. It was not possible to use sufficient replication of machine type in each laboratory to examine all sources of variation in this study, but the results strongly indicate that factors other than machine type or manufacturer (e.g., method or laboratory) contribute more to variation in a GM quantification result.     

Four-stranded DNA structure stabilized by a novel G:C:A:T tetrad

The solution structure of a cyclic oligonucleotide d has been determined by two-dimensional NMR spectroscopy and restrained molecular dynamics. Under the appropriate experimental conditions, this molecule self-associates, forming a symmetric dimer stabilized by four intermolecular Watson-Crick base pairs. The resulting four-stranded structure consists of two G:C:A:T tetrads, formed by facing the minor groove side of the Watson-Crick base-pairs. Most probably, the association of the base-pairs is stabilized by coordinating a Na(+) cation. This is the first time that this novel G:C:A:T tetrad has been found in an oligonucleotide structure. This observation increases considerably the number of sequences that may adopt a four-stranded architecture. Overall, the three-dimensional structure is similar to those observed previously in other quadruplexes formed by minor groove alignment of Watson-Crick base pairs. This resemblance strongly suggests that we may be observing a general motif for DNA-DNA recognition.     

Discrimination of A1555G and C1494T point mutations in the mitochondrial 12S rRNA gene by on/off switch

The objective of this study was to apply the “on/off” switch consisting of 3′ phosphorothioate-modified allele specific primers and exo⁺ polymerase in single base discrimination of A1555G and C1494T mutations in the highly conserved sites of the mitochondrial 12S rRNA. The two point mutations are the hotspot mutations associated with either aminoglycoside antibiotics induced deafness or inherited nonsyndromic hearing loss. The PCR products of mitochondrial DNA (mtDNA) 12S rRNA gene were inserted into the pMD19-T vector for transformation into Escherichia coli JM109 competent cells for preparing wild-type pMD19-T/mt vector. Inverse PCR was carried out for mtDNA 12S rRNA gene C1494T and A1555G mutagenesis and DpnI endonuclease degradating methylated pMD19-T/mt vector existing in the inverse PCR products was carried out to construct the mutation-type pMD19-T/mtM vector. These constructed vectors were confirmed by DNA sequencing. Allelic specific primers targeting wild-type and mutation-type templates were designed with 3′ terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases. Amplified by exo⁺ polymerase, allelic specific primers perfectly matching wild-type allele were extended while no products were produced from primers targeting point-mutated deafness-related allele. Similarly, allelic specific primers perfectly matching point-mutated deafness-related mutation-type allele were extended and no products were yielded from primers targeting wild-type allele. No specific product was observed in the primer extension reaction mediated by on/off switch in screening the mtDNA 12S rRNA gene harboring either C1494T or A1555G mutation in 40 healthy volunteers tested. These data suggest that the “off switch” mediated by exo⁺ polymerase is highly reliable in the diagnosis of monogenic diseases and the novel “on/off” switch has enormous applications in systematic and extended screening of the12S rRNA gene A1555G and C1494T mutations. The established assay can be widely used not only for hearing loss patients but also for normal subjects before the use of aminoglycoside antibiotics.     

A fluorescence-based sequence-specific primer PCR for the screening of HLA-B(*)57:01

Abacavir (ABC) is an antiretroviral drug highly effective in the treatment of HIV, but its intake can cause severe hypersensitivity reaction (HSR). A strong association between HLA-B(*)57:01 and ABC HSRs was reported by several studies, which demonstrated that HLA-B(*)57:01 screening had a 100% negative predictive value and that it could accurately identify patients at high risk of ABC HSRs. We propose a new sequence-specific primer PCR assay based on fluorescence detection through CE which is highly sensitive, allowing the use of non-infective sources of DNA such as saliva and buccal swabs, in addition to blood and reproducible, allowing automation of the analytical process. The results of our study were first compared with a standard sequence-specific primer PCR technique and reported a concordance of 100%, and then a blind external validation further confirmed the accuracy of our method.     

Capillary and microchip gel electrophoresis for simultaneous detection of Salmonella pullorum and Salmonella gallinarum by rfbS allele-specific PCR

We report the use of capillary gel electrophoresis (CGE) based on a rfbS allele-specific polymerase chain reaction (PCR) for the analysis and simultaneous detection of Salmonella pullorum and Salmonella gallinarum, which are the major bacterial pathogens in poultry. rfbS allele-specific PCR was used to concurrently amplify two specific 147- and 187-bp DNA fragments for the simultaneous detection of S. pullorum and S. gallinarum at an annealing temperature of 54 ± 1 °C and an MgCl₂ concentration of 2.8-5.6 mM. Under an electric field of 333.3 V/cm and a sieving matrix of 1.0% poly(ethyleneoxide) (M_r 600 000), the amplified PCR products were analyzed within 6 min by CGE separation. This CGE assay could be translated to microchip format using programmed field strength gradients (PFSG). In the microchip gel electrophoresis with PFSG, both of the Salmonella analyses were completed within 30 s, without decreasing the resolution efficiency. rfbS allele-specific PCR-microchip gel electrophoresis with the PFSG technique might be a new tool for the simultaneous detection of both S. pullorum and S. gallinarum, due to its ultra-speed and high efficiency.     

Tuning single nucleotide discrimination in polymerase chain reactions (PCRs): synthesis of primer probes bearing polar 4'-C-modifications and their application in allele-specific PCR

There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele-specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR-amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes by using allele-specific primer probes. PCR amplification by a DNA polymerase from matched 3'-primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real-time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4'-C-modified nucleotide residues at their 3' termini, and their evaluation in real-time asPCR. We found that primer probes bearing a 4'-C-methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.     

An analogue of adenine that forms an "A:T" base pair of comparable stability to G:C

The heterocyclic base 7-aminopropargyl-7-deaza-2,6-diaminopurine (D) has been incorporated into oligodeoxynucleotides. D:T has similar thermodynamic stability to G:C and is a stable analogue of A:T.     

Label-free detection of DNA mutations by SPR: application to the early detection of inherited breast cancer

A screening analysis of DNA hybridization and the presence of DNA mutations using an surface plasmon resonance (SPR) biosensor is shown. The influence of lateral and vertical spacers, as well as several hybridization conditions, was studied to optimize the differentiation between fully complementary and mismatched DNA strands. Our results demonstrated that SPR biosensors were able to detect mismatch sequences related to inherited breast cancer, with high specificity and sensitivity. Using PCR synthetic sequences as targets, mutant sequences were clearly discriminated from fully complementary ones, and detection limits below 50 nM were achieved.     

Stabilization of fluorescent silver clusters by RNA homopolymers and their DNA analogs: C,G versus A,T(U) dichotomy

Single-stranded RNAs host fluorescent silver nanoclusters, analogous to previously studied Ag:DNAs. C and G homopolymers stabilize visible- to IR-emitting silver complexes at neutral pH in both RNA and DNA, but A and T/U do not. Cluster fluorescence is sensitive to sugar as well as base type.     

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.     

Direct modification of mRNA by ferrocenyl carbodiimide and its application to electrochemical detection of mRNA.

Ferrocenyl carbodiimide (1) could be used for the direct labeling of synthetic RNA and expressed mRNA in vitro with the electrochemically active ferrocene moieties. These RNAs modified by 1 could be detected electrochemically coupled with a DNA probe-immobilized electrode. After hybridization of 1.1 Kb mRNA modified by 1 with the DNA probe-immobilized electrode, the peak charge observed by an Osteryoung square wave voltammetry (SWV) measurement correlated well with the concentration of mRNA, having a detection limit at the sub nanogram level.     

MP2 study on the hydrogen-bonding interaction between 5-fluorouracil and DNA bases: A,C,G,T

The 5-fluorouracil is a pyrimidine analog effective in the treatment of cancer. In this work, we present the hydrogen-bonding base pairs involving 5-FU bound to the four bases in DNA: adenine, cytosine, guanine, and thymine. Full geometry optimizations have been performed for the studied complexes by MP2 method. The interaction energies were corrected for the basis-set superposition error, using the full Boys-Bernardi counterpoise correction scheme. Hydrogen-bonding patterns of these base pairs were characterized using NBO analysis and AIM analysis. According to the calculated binding energies and structural parameters, the stability of the base pairs decrease in the following order: 5-FU:A > 5-FU:G > 5-FU:T > 5-FU:C.     

MP2 study on the hydrogen-bonding interaction between 5-hydroxymethyl-uracil and DNA bases: A,C, G,T

The 5-hydroxymethyl-uracil (HmU) is a product of oxidative attack on the methyl group of thymine in DNA. In this work, we present the hydrogen bonding complexes formation involving HmU bound to the four bases in DNA: adenine (A), cytosine (C), guanine (G), and thymine (T). Full geometry optimizations have been performed for the studied complexes by MP2 method. The interaction energies were corrected for the basis-set superposition error (BSSE), using the full Boys-Bernardi counterpoise correction scheme. Hydrogen bonding patterns of these base pairs were characterized using NBO analysis and AIM analysis. According to the calculated binding energies and structural parameters, the stability of the base pairs decrease in the following order: HmU:A > HmU:G > HmU:C > HmU:T.     

MP2 study on the hydrogen-bonding interaction between 5-fluorocytosine and DNA bases: A,C, G,T

The 5-fluorocytosine (5-FC) is a fluorinated cytosine analog that is used as an antifungal agent. In this work, we present the hydrogen-bonding base pairs involving 5-FC bound to the four bases in DNA: adenine (A), cytosine (C), guanine (G), and thymine (T). Full geometry optimizations have been performed for the studied complexes by MP2 method. The interaction energies were corrected for the basis set superposition error, using the full Boys–Bernardi counterpoise correction scheme. Hydrogen-bonding patterns of these base pairs were characterized using NBO analysis and AIM analysis. According to the calculated binding energies and structural parameters, the stability of the base pairs decreases in the following order: 5-FC:G > 5-FC:C > 5-FC:A > 5-FC:T.     

A multifaceted phosphate tether: application to the C15-C30 subunit of dolabelides A-D

Construction of the C15-C30 subunit of dolabelide utilizing a temporary phosphate tether is described. Two routes are reported that make use of the orthogonal protecting- and leaving-group properties innate to phosphate esters. One route relies on a selective terminal oxidation, while a second utilizes a CM/selective hydrogenation sequence. Both routes depend on a highly regio- and diastereoselective cuprate addition to set the requisite stereochemistry at C22.     

The mechanism of the hantzsch pyridine synthesis: A study by 15N and 13C NMR spectroscopy

The mechanism of the reactions of ammonia and benzaldehyde with three different beta-dicarbonyl compounds to form the corresponding dihyropyridines has been followed by NMR. In each case the pathway is shown to involve the reaction of benzaldehyde with one molecule of beta-dicarbonyl to give chalcone, and of the ammonia with a second molecule of beta-dicarbonyl to give an enamine. The rate determining stage is shown to be the Michael addition of the chalcone to the enamine.     

Immobilisation of quantum dots by bio-orthogonal PCR amplification and labelling for direct gene detection and quantitation

A sensitive and versatile detection scheme based on quantum dot immobilisation on a solid support through bio-orthogonal PCR amplification and labelling has been developed for detection and quantification of gene targets in complex DNA mixtures.     

MP2 study on the hydrogen-bonding interaction between O 4-methylthymine and DNA bases: A,C, G,and T

The O ⁴-methylthymine (m⁴T) is a nucleobase lesion induced by the action of ionizing radiation on thymine residue in DNA. In this study, we present the hydrogen-bonding base pairs involving m⁴T bound to the four bases in DNA: adenine (A), cytosine (C), guanine (G), and thymine (T). Full geometry optimizations have been performed for the studied complexes by MP2 method. The interaction energies were corrected for the basis-set superposition error, using the full Boys–Bernardi counterpoise correction scheme. Hydrogen-bonding patterns of these base pairs were characterized using NBO analysis and AIM analysis. According to the calculated binding energies and structural parameters, the stability of the base pairs decrease in the following order: m⁴T:G > m⁴T:A > m⁴T:C > m⁴T:T.