Asymmetric Polymerase Chain Reaction Amplification Research Articles

A Study of the Detection of SARS-CoV-2 by the Use of Electrochemiluminescent Biosensor Based on Asymmetric Polymerase Chain Reaction Amplification Strategy.

A new and reliable method has been constructed for detecting severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) open reading frames 1ab (ORF1ab) gene via highly sensitive electrochemiluminescence (ECL) biosensor technology based on highly efficient asymmetric polymerase chain reaction (asymmetric PCR) amplification strategy. This method uses magnetic particles coupled with biotin-labeled one complementary nucleic acid sequence of the SARS-CoV-2 ORF1ab gene as the magnetic capture probes, and [Formula: see text]-labeled amino-modified another complementary nucleic acid sequence as the luminescent probes, and then a detection model of magnetic capture probes-asymmetric PCR amplification nucleic acid products-[Formula: see text]-labeled luminescent probes is formed, which combines the advantages of highly efficient asymmetric PCR amplification strategy and highly sensitive ECL biosensor technology, enhancing the method sensitivity of detecting the SARS-CoV-2 ORF1ab gene. The method enables the rapid and sensitive detection of the ORF1ab gene and has a linear range of 1-[Formula: see text] copies/[Formula: see text], a regression equation of [Formula: see text] = [Formula: see text] + 2919.301 ([Formula: see text] = 0.9983, [Formula: see text] = 7), and a limit of detection (LOD) of 1 copy/[Formula: see text]. In summary, it can meet the analytical requirements for simulated saliva and urine samples and has the benefits of easy operation, reasonable reproducibility, high sensitivity, and anti-interference abilities, which can provide a reference for developing efficient field detection methods for SARS-CoV-2.

Development and Clinical Evaluation of Oligonucleotide Microarray for HLA-AB Genotyping

The antigens encoded by human leukocyte antigen (HLA) genes are primary antigens in immunological response of transplantation, and genotypes of HLA-A, B and DRB1 must be determined on donors and recipients before the transplantation is carried out. In this study, oligonucleotide microarray for HLA-AB genotyping was prepared and evaluated. Oligonucleotide probes were designed based on the sequence of the different genotypes of HLA-A and B and were fixed on silylated glass slides to form a microarray. Fluorescence-labeled target DNA was obtained by asymmetric polymerase chain reaction amplification of exon 2 and exon 3 of HLA-A and -B genes and hybridized to the microarray. The hybridized microarray was subsequently scanned and the result was analyzed by software in order to determine the genotypes of the tested sample. The sensitivity of the microarray for HLA-AB genotyping was 10 ng/microl, with a coincidence rate of 100% with international reference, and a combined variation value of detected signal below 15%. Analysis of genotyping of 1295 cases of clinical samples showed that the general coincidence rate between the microarray method and conventional method (polymerase chain reaction-sequence-specific primer and flow cytometry reverse polymerase chain reaction sequence-specific oligonucleotide) was HLA-A: 99.61% and HLA-B: 98.30%, respectively. A total of five out of seven samples that had conflicting results of genotypes were proved to be microarray-assay reliable by DNA sequencing, suggesting a higher accuracy of the microarray method. The microarray for HLA-AB genotyping is satisfactory for clinical use in HLA-AB genotyping with its good specificity, sensitivity and reproducibility.

Combination of amplification and post-amplification strategies to improve optical DNA sensing

The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX™). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection.

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 microl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.

Allele-specific and asymmetric polymerase chain reaction amplification in combination: A one step polymerase chain reaction protocol for rapid diagnosis of familial defective apolipoprotein B-100

We have combined the asymetric polymerase chain reaction (PCR) with allele-specific PCR to detect a single point mutation. A set of two priming oligonucleotides and a third allele-specific primer were used to identify heterozygotes for a G to A mutation at nucleotide 10,708 in the apolipoprotein B (apo B) gene. The system requires neither restriction enzyme digestion nor allele-specific oligonucleotides as conventionally applied for allele-specific hybridization of slot blots. This method clearly allows for the detection of the mutant allele by inspection, after agarose gel electrophoresis of a single PCR reaction. DNA from 40 patients with familial defective apo B-100 due to the G to A mutation at nucleotide 10,708 in the apo B gene and their normal relatives was analyzed. Complete agreement with allele-specific hybridization of slot blots confirms supposition that the system is effective to screen a larger population.

Molecular Basis for Antithrombin III Type I Deficiency: Three Novel Mutations Located in Exon IV

AbstractAntithrombin III (AT III) type I deficiencies are characterized by a 50% decrease of both immunoreactive and functional protein and carry a high risk of thrombotic complication. We have studied the molecular basis for such deficiencies by asymmetric polymerase chain reaction amplification and direct sequencing of the seven exons and of the intron-exon junction of the AT III gene. Three different mutations were observed in the exon IV: a 4-bp deletion, a 2-bp deletion, and a nucleotide insertion. Each of these mutations results in a frameshift introducing premature stop codons at positions 313, 309, and 232, respectively. These results were confirmed by dot-blot analysis with allele-specific oligonucleotide probes. Furthermore, no mutation was observed in the other six exons. The comparison of the type of mutations observed by our group in six cases of type I deficiencies and in 16 cases of type II heparin binding site variants deficiencies suggests that the former are caused by heterogeneous molecular abnormalities while the latter are caused by recurrent mis-sense mutations

Molecular basis for antithrombin III type I deficiency: three novel mutations located in exon IV

Antithrombin III (AT III) type I deficiencies are characterized by a 50% decrease of both immunoreactive and functional protein and carry a high risk of thrombotic complication. We have studied the molecular basis for such deficiencies by asymmetric polymerase chain reaction amplification and direct sequencing of the seven exons and of the intron-exon junction of the AT III gene. Three different mutations were observed in the exon IV: a 4-bp deletion, a 2-bp deletion, and a nucleotide insertion. Each of these mutations results in a frameshift introducing premature stop codons at positions 313, 309, and 232, respectively. These results were confirmed by dot-blot analysis with allele-specific oligonucleotide probes. Furthermore, no mutation was observed in the other six exons. The comparison of the type of mutations observed by our group in six cases of type I deficiencies and in 16 cases of type II heparin binding site variants deficiencies suggests that the former are caused by heterogeneous molecular abnormalities while the latter are caused by recurrent missense mutations.