Chain Amplification Reaction Research Articles

Peripheral substitution effects on unnatural base pairs: A case of brominated TPT3 to enhance replication fidelity

The high fidelity poses a central role in developing unnatural base pairs (UBPs), which means the high pairing capacity of unnatural bases with their partners, and low mispairing with all the natural bases. Different strategies have been used to develop higher-fidelity UBPs, including optimizing hydrophobic interaction forces between UBPs. Variant substituent groups are allowed to fine tune the hydrophobic forces of different UBPs’ candidates. However, the modifications on the skeleton of TPT3 base are rare and the replication fidelity of TPT3-NaM remains hardly to improve so far. In this paper, we reasoned that modifying and/or expanding the aromatic surface by Bromo-substituents to slightly increase hydrophobicity of TPT3 might offer a way to increase the fidelity of this pair. Based on the hypothesis, we synthesized the bromine substituted TPT3, 2-bromo-TPT3 and 2, 4-dibromo-TPT3 as the new TPT3 analogs. While the enzyme reaction kinetic experiments showed that d2-bromo-TPT3-dNaM pair and d2, 4-dibromo-TPT3TP-dNaM pair had slightly less efficient incorporation and extension rates than that of dTPT3-dNaM pair, the assays did reveal that the mispairing of 2-bromo-TPT3 and 2, 4-dibromo-TPT3 with all the natural bases could dramatically decrease in contrast to TPT3. Their lower mispairing capacity promoted us to run polymerase chain amplification reactions, and a higher fidelity of d2-bromo-TPT3-dNaM pair could be obtained with 99.72 ± 0.01% of the in vitro replication fidelity than that of dTPT3-dNaM pair, 99.52 ± 0.09%. In addition, d2-bromo-TPT3-dNaM can also be effectively copied in E. coli cells, which showed the same replication fidelity as that of dTPT3-dNaM in the specific sequence, but a higher fidelity in the random sequence context.

Electrochemical DNA Biosensors with Dual-Signal Amplification Strategy for Highly Sensitive HPV 16 Detection.

Cervical cancer is an important topic in the study of global health issues, ranking fourth among women's cancer cases in the world. It is one of the nine major cancers that China is focusing on preventing and treating, and it is the only cancer that can be prevented through vaccination. Systematic and effective screening for human papilloma (HPV) infection, which is closely linked to the development of cervical cancer, can reduce cervical cancer incidence and mortality. In this paper, an electrochemical sensor was designed to detect HPV 16 using dual-signal amplification. An APTES-modified glassy carbon electrode was used for improved stability. Gold nanoparticles and a chain amplification reaction were combined for signal amplification. The limit of detection (LOD) of this electrochemical sensor was 1.731 × 10-16 mol/L, and the linear response of the target detector range was from 1.0 × 10-13 mol/L to 1.0 × 10-5 mol/L (R2 = 0.99232). The test of serum sample recovery showed that it has good anti-interference, and the performance of all aspects was improved to different degrees compared with the previous research from the team. The designed sensor is centered around the principles of low cost, high sensitivity and stability, which provides new ideas for the future development of cervical cancer prevention and electrochemical biosensors.

Intelligent dual-drive DNA nanosensor for ultrasensitive detection of prostate cancer-related circulating microRNA-200c

Rapid and accurate detection of the dynamic changes of prostate cancer-related miRNAs in peripheral blood is of great significance for prostate monitoring. Herein, we constructed an intelligent dual-drive DNA nanosensor (DD-DNS) for molecularly determination of microRNA-200c (miR-200c), a potential monitoring marker for diagnosis and treatment of prostate cancer. Unlike common detection technologies, the most distinctive feature of DD-DNS is the ingenious combination of enzyme-catalyzed chain amplification reaction and non-enzyme-catalyzed chain replacement reaction to realize the complementation of performance and improvement of detection capability. This improvement can significantly improve the detection sensitivity and environmental adaptability, which is more conducive to the detection of actual samples. In the presence of miR-200c, miR-200c activates the polymerase/nickase cycle reaction, resulting in exponential levels of cycle primers being produced. As a programmed process, cyclic primers catalyze the hairpin assembly reaction to achieve a surge in fluorescence signals. As a novel amplification strategy, the detection limit of this method can reach 5.5 fM, which fully meets the requirements of clinical quantitative analysis. Thus, miRNA detection technology based on DD-DNS shows superior detection performance and stability, which is conducive to carrying out extensive disease screening and treatment monitoring in a variety of complex situations.

Polymorphisms of genes of the renin-angiotensin system: significance in the progression of chronic kidney disease in children

Introduction. A comprehensive analysis of the polymorphisms of the genes of the renin-angiotensin system in children with different nosological forms of nephropathies is a necessary step in determining the clinical and genetic features of the formation of chronic kidney disease (CKD). 
 Aim: to establish the features of ACE (D/I), GT (Thr174Met), AGT (Met235Thr) and AGTR1 (A1166C) gene polymorphisms in CKD children and determine their significance in the progression of the disease. 
 Materials and methods. A retrospective and prospective study included one hundered 1 to 17 years children with nephropathies, examined in the children’s nephrology department of the Samara Regional Hospital over 10 years. In children, the identification of single nucleotide polymorphisms of genes was carried out using an allele-specific polymerase chain amplification reaction using test systems. Evaluation of clinical and paraclinical markers of progression in CKD was carried out twice a year. The results of the study were evaluated with the calculation of the Student–Fisher criteria and correlation analysis.
 Results. in patients with kidney diseases, there was a trend towards an increase in the occurrence of single nucleotide polymorphisms of genes that affect the renin-angiotensin system (RAS). CKD patients at the stage 3–5 accounted for 35%. They had D/D ACE alleles combined with alleles AGT Thr174Met (27.9 ± 6.83%) and Met235Thr (41.86 ± 7.5%), Thr235Thr (30.2 ± 7.0%) and AGTR1 A1166C (37.2 ± 7.32%) more often than in milder CKD (0 and 7.5 ± 3.37%; 5.2 ± 2.94% and 5.2 ± 2.94%; respectively, p ≤ 0.010). 
 Соnclusion. The study of clinical and genetic features of CKD is relevant for the purpose of timely implementation of preventive measures.

Ultrasensitive isothermal method to detect microRNA based on target-induced chain amplification reaction

We herein describe an ultrasensitive isothermal method to detect microRNA (miRNA) by utilizing target-induced chain amplification reaction (CAR). The hairpin probe (HP) employed in this strategy is designed to be opened upon binding to target miRNA. The exponential amplification reaction (EXPAR) template (ET) then binds to the exposed stem of HP and DNA polymerase (DP) promotes the extension reactions for both HP and ET, consequently producing intermediate double-stranded DNA product (IP) and concomitantly recycling target miRNA to open another intact HP. The IPs would produce a large number of target-mimicking probes (TMPs) and trigger probes (TPs) through the continuously repeated nicking and extension reactions at the two separated nicking sites within the IP. TMP triggers another CAR cycle by binding to intact HP as target miRNA did while TP promotes conventional EXPAR by independently binding to free ET. As a consequence of these interconnected reaction systems, a large number of final double-stranded DNA products (FPs) are produced, which can be monitored by measuring the fluorescent signal produced from duplex-specific fluorescent dye. Based on this unique design principle, the target miRNA was successfully determined down to even a single copy with high selectivity against non-specific miRNAs. The practical applicability of this method was also verified by reliably detecting target miRNA included in the total RNA extracted from the human cancer cell.

Aptamer-Capped nanoporous anodic alumina for Staphylococcus aureus detection

The development of new detection systems for an accurate and rapid identification of pathogens has become an essential challenge in the biomedical field. Herein a highly selective platform based on aptamer-gated nanomaterials for specific Staphylococcus aureus detection is presented. In the proposed design, a nanoporous anodic alumina (NAA) scaffold is loaded with the fluorescent indicator rhodamine B, while pores entrances are capped by a DNA aptamer which selectively recognizes S. aureus cells in less than 1 h. When S. aureus cells are present, the solid is selectively uncapped, and the dye is released to the medium. This nanodevice allows the detection of bacterial concentrations between 2 and 5 CFU mL−1 (in buffer and blood, respectively) and it has demonstrated excellent behavior in terms of specificity and robustness. A set of 25 different clinical samples are analyzed using this simple procedure obtaining excellent results, which agree with conventional hospital reference techniques for the identification of S. aureus. This new method is sensitive, rapid and low cost, and avoids steps such as polymerase chain amplification reaction, which makes it suitable for use in point-of-care detection systems.

Self-assembled poly-HRP dual signal amplification strategy for high-sensitive detection of circulating miR-142-3p in human serum

The establishment of a highly sensitive and efficient platform for detecting circulating miRNA in serum is important for disease diagnosis and prognosis. To this end, a self-assembled horseradish peroxidase (HRP)-based polymer (poly-HRP) dual signal amplification strategy, via skillful combination of functionalized magnetic nanoparticles as the capture nanoprobes and the poly-HRP as signal amplification probes, has been developed for highl-sensitive and accurate detection of circulating miR-142-3p in human serum. Just by mixing building blocks together at one time, the poly-HRP used for dual signal amplification can be engineered by only one-step assembly. As the poly-HRP can be directly prepared ahead, avoiding chain amplification reaction in the detection process, the assay can be rapidly accomplished in about one hour. The measured limit-of-detection (LOD) is down to 100 fM. Standard miR-142-3p spiked in human serum is measured with ideal recovery rates ranging from 97.46% to 115.90%. More importantly, this method was validated to be applicable to direct detection of miR-142-3p in serums, and the results suggested that the miR-142-3p levels of serum samples obtained from T-cell acute lymphoblastic leukemia children (n = 25) are significantly higher than those from healthy control groups (n = 25) (P < 0.01). To conclude, the ingenious combination of functionalized magnetic nanoparticles and the poly-HRP presented in this work brings forth a rapid, convenient, yet simple, sensitive, and specific biosensing method that can potentially be applied for clinical diagnosis.

Prenatal diagnosis of Larsen syndrome caused by a mutation in the filamin B gene

Prenatal diagnosis of Larsen syndrome caused by a mutation in the filamin B gene

Principal component analysis and discriminant analysis (PCA–DA) for discriminating profiles of terminal restriction fragment length polymorphism (T-RFLP) in soil bacterial communities

To assess the impact of a transgenic crop on soil environment, we compared soil bacterial communities from the rhizospheres of cucumber green mottle mosaic virus (CGMMV)-resistant transgenic watermelon ( Citrullus vulgaris [Twinser] cv. Gongdae) and non-transgenic parental line watermelon at an experimental farm in Miryang, Korea. Soil microbial community structure was studied using terminal restriction fragment length polymorphism (T-RFLP) using HaeIII and HhaI enzymes on products from polymerase chain amplification reactions (PCR) of total DNA from rhizosphere. We used principal component analyses (PCA) to reduce dimensionality of T-RFLP profiles before comparison. On these PCA scores, we conducted discrimination analyses to compare soil microbial communities from the rhizosphere of transgenic and non-transgenic. Discriminant analyses indicate that microbial communities from rhizosphere of transgenic and non-transgenic watermelon did not differ with significance at 95% level. Our study could be used as a model case to assess the environmental risk assessment of transgenic crops on soil microbial organisms.

Some coat protein constituents from strawberry latent ringspot virus expressed in transgenic tobacco protect plants against systematic invasion following root inoculation by nematode vectors

The coding sequences in RNA2 for the coat proteins (CP) of strawberry latent ringspot virus (SLRSV) were modified and amplified using polymerase chain amplification reactions (PCR) to facilitate their expression inAgrobacterium tumefaciens-transformedNicotiana tabacum Xanthi-nc. The coding sequences for the smaller capsid protein (S, 29kDa) and that for the theoretical precursor of L and S (P, 73kDa) had ATG ‘initiation’ codon sequences added at the 5′-proximal Ser/Gly (S/G) cleavage site in the unmodified sequence. The sequence coding for the larger of the two proteins of mature SLRSV capsids (L, 44kDa) had an ATG codon added at its 5′ S/G site and a TAG ‘stop’ codon sequence added at the 3′-proximal S/G site. The P, L and S proteins were expressedin planta to a maximum concentration of 0.01 % of total extractable proteins but did not assemble into virus-like particles. When challenged by mechanical inoculation with virus particles or viral RNA, and compared with control plants, tobacco plants (primary transgenic clones or S1 and S2, kanamycin-resistant seedlings) expressing the virus capsid subunits separately, or their precursor, decreased the accumulation of SLRSV particles in inoculated leaves and fewer plants became invaded systemically. In experiments in which the roots of seedlings were exposed to SLRSV-carrying vector nematodes (Xiphinema diversicaudatum), SLRSV was detected in the roots of non-transformed control tobacco plants (6/20) and in transgenic tobacco expressing the L protein (7/40), but not in any of 25 tobacco plants expressing the S protein or in 35 expressing the P protein. This is the second example of CP-mediated resistance to virus inoculation by nematode vectors.

Alterations in spliced and unspliced HIV-1-specific RNA detection in peripheral blood mononuclear cells of individuals with varying CD4-positive lymphocyte counts.

Reverse transcriptase-polymerase chain amplification reactions (RT-PCR) were used to identify transcripts for HIV-1 structural and regulatory proteins in peripheral blood mononuclear cells of a cohort of 48 patients. At least one set of PCR primers was capable of detecting HIV-1 transcripts in 94% of patients. Unspliced gag-pol transcripts were detected with gag or pol primer sets in 60 and 63% of samples, respectively. A significant inverse correlation was noted between transcript identification with the gag primer set and the number of CD4-positive lymphocytes in the blood sample and the clinical stage of infection. Single-spliced env transcripts were identified in 44% of individuals. Multiple-spliced tat or nef transcripts were detected in 6.2 and 53% of individuals, respectively. These findings indicate that viral transcripts are expressed throughout the course of HIV-1 infection.

Polymerase chain reaction amplification of single-stranded DNA containing a base analog, 2-chloroadenine

By utilization of polymerase chain reaction techniques, single-stranded DNA of defined length and sequence containing a purine analog, 2-chloroadenine, in place of adenine was synthesized. This was accomplished by a combination of standard polymerase chain amplification reactions with Thermus aquaticus DNA polymerase in the presence of four normal deoxynucleoside triphosphates, M13 duplex DNA as template, and two primers to generate double-stranded DNA 118 bases in length. An asymmetric polymerase chain reaction, which produced an excess of single-stranded 98-base DNA, was then conducted with 2-chloro-2′-deoxyadenosine 5′-triphosphate in place of dATP and with only one primer that annealed internal to the original two primers. Standard polymerase chain reaction techniques alone conducted in the presence of the analog as the fourth nucleotide did not produce duplex DNA that was modified within both strands. This asymmetric technique allows the incorporation of an altered nucleotide at specific sites into large quantities of single-stranded DNA without using chemical phosphoramidite synthesis procedures and circumvents the apparent inability of DNA polymerase to synthesize fully substituted double-stranded DNA during standard amplification reactions. The described method will permit the study of the effects of modified bases in template DNA on a variety of protein-DNA interactions and enzymes.

Temporal regulation of viral and cellular gene expression during human T-lymphotropic virus type I-mediated lymphocyte immortalization

An autocrine mechanism of proliferation may play a significant role in the leukemogenesis of adult T-cell leukemia, a mature T-cell malignancy caused by human T-lymphotropic virus type I (HTLV-I). To further delineate the role of HTLV-I and the interleukin 2 (IL2) system in the transformation process, human primary lymphocytes were infected by cocultivation with lethally X-irradiated MT2 cells in the presence or absence of human rIL2; the polymerase chain amplification reaction was used to examine quantitatively the expression of HTLV-I, IL2, and IL2R alpha mRNAs during early and late proliferation phases that displayed polyclonal (days 7 to 49) and oligoclonal (days 100 to 150) proviral integration, respectively. IL2 mRNA and IL2 activity were transiently expressed during the polyclonal phase but were undetectable at later time points. IL2R alpha mRNA expression remained at a constitutively high level throughout the examined time course. Viral transcripts were detectable at each time point. Expression of the tax-rex mRNA was inversely related to IL2 mRNA levels; it was low early in the polyclonal phase but increased 30-fold with the development of oligoclonality. In addition, paraformaldehyde-fixed HTLV-I-producing cells activated peripheral blood lymphocytes. These data suggest that HTLV-I activates human T lymphocytes. However, IL2 expression is transient, indicating a limited involvement of an IL2 autocrine growth loop in the transformation process. Lastly, another viral determinant, in addition to the trans activator tax, may be important in HTLV-I-induced T-cell proliferation.

The application of oligonucleotide probes to HLA Class II typing of the DRB sub‐region

A plan for DRB typing at the sequence level is detailed. Only one polymerase chain amplification reaction is needed and the application of a limited number of short oligonucleotide probes allows an almost complete definition of DRB alleles. The scheme was tested on 40 homozygous cell-lines selected to cover a wide range of specificities, and 40 RFLP-typed controls. The results are presented and discussed. The simplicity and accuracy of this scheme are emphasized.

Specificity of polymerase chain amplification reactions for human immunodeficiency virus type 1 DNA sequences.

The polymerase chain amplification reaction (PCR) is a sensitive, specific, and quantitative assay of human immunodeficiency virus type 1 (HIV-1). The assay was performed with polymerases from Escherichia coli or Thermus aquaticus (Taq). A single pair of oligonucleotide primers within the long terminal repeat (LTR) sequences were used to detect HIV-1 sequences in infected cell cultures and fresh tissues of the large majority of infected individuals. The amplified product was a faithful copy of this LTR sequence. Utilization of a subsaturating number of cycles of amplification allowed quantitation of HIV-1 DNA sequences.