Exploring of tri-allelic SNPs using pyrosequencing and the SNaPshot methods for forensic application

Tri-allelic single nucleotide polymorphisms (SNPs) are potential forensic markers for DNA analysis. Currently, only a limited number of tri-allelic SNP loci have been proved to be fit for forensic application. In this study, we aimed to develop an effective method to select and genotype tri-allelic SNPs based on both Pyrosequencing (PSQ) and the SNaPshot methods. 50 candidate SNPs were chosen from NCBI's dbSNP database and were analyzed by PSQ. The results revealed that 20 SNPs were tri-allelic and were located on 16 autosomal chromosomes. Then 20 SNP loci were combined in one multiplex polymerase chain reaction to develop a single base extension (SBE)-based SNP-typing assay. A total of 100 unrelated Chinese individuals were genotyped by this assay and allele frequencies were estimated. The total discrimination power was 0.999999999975 and the cumulative probability of exclusion was 0.9937. These data demonstrated that the strategy is a rapid and effective method for seeking and typing tri-allelic SNPs. In addition, the 20 tri-allelic SNP multiplex typing assay may be used to supplement paternity testing and human identification.     

Efficient Discrimination of Single Nucleotide Polymorphisms (SNPs) Using Oligonucleotides Modified with C5‐Pyrene‐Functionalized DNA and Flanking Locked Nucleic Acid (LNA) Monomers

Oligodeoxyribonucleotides modified with 5‐[3‐(1‐pyrenecarboxamido)propynyl]‐2′‐deoxyuridine monomer X and proximal LNA monomers display higher affinity for complementary DNA, more pronounced increases in fluorescence emission upon DNA binding, and improved discrimination of SNPs at non‐stringent conditions, relative to the corresponding LNA‐free probes across a range of sequence contexts. The results reported herein suggest that the introduction of LNA monomers influences the position of nearby fluorophores via indirect conformational restriction, a characteristic that can be utilized to develop optimized fluorophore‐labeled probes for SNP‐discrimination studies.     

Fluorescence Detection of Single‐Nucleotide Polymorphisms with a Single,Self‐Complementary,Triple‐Stem DNA Probe

Singled out for its singularity : In a single‐step, single‐component, fluorescence‐based method for the detection of single‐nucleotide polymorphisms at room temperature, the sensor is comprised of a single, self‐complementary DNA strand that forms a triple‐stem structure. The large conformational change that occurs upon binding to perfectly matched (PM) targets results in a significant increase in fluorescence (see picture; F=fluorophore, Q=quencher).

     

Resolving human DNA mixtures with F‐108 polymer and capillary electrophoresis single‐strand conformational polymorphism analysis

Current technologies have increased the sensitivity for analyzing forensic DNA samples, especially those considered “touch samples.” Because of this, there has been an increase in the number of forensic mixtures–two or more contributors within a single sample–submitted to the crime laboratories. Therefore, the need to resolve these mixtures has increased as well. Several technologies are currently utilized, but many of them are time consuming and do not resolve the entire profile. Therefore, CE‐Single‐Strand Conformational Polymorphisms coupled with the Pluronic F‐108 polymer was assessed for its ability to resolve human forensic mixtures. This technique has been able to detect sequence variation, such as single nucleotide polymorphism in short tandem repeat loci, such as D7S820 and vWA. Samples were first analyzed with the Performance Optimized Polymer‐7, and mixtures created from samples that shared alleles. These samples were sequenced to detect single base‐pair mutations and evaluated with the F‐108 and CE‐Single Strand Conformational Polymorphism analysis. Results from this study indicated the method would serve as a valuable screening tool to detect base sequence variation between individuals when they share alleles in a mixture and before using Massive Parallel Sequencing technology to distinguish which bases differ.     

Laboratorial diagnosis of paracoccidioidomycosis and new insights for the future of fungal diagnosis

Paracoccidioidomycosis (PCM) is the most prevalent mycosis in Latin-America. As for other mycosis, its importance of has been largely underestimated, partially due to the limited geographical distribution of the etiologic fungal agent (Paracoccidioides brasiliensis). However, the advent of AIDS and other immune suppressing conditions is creating an emergent need for improved diagnostic tests envisaging simpler, cheaper, faster and more sensitive and accurate detection of pathogenic fungi, especially those causing systemic and opportunistic diseases. Routine laboratorial diagnosis of PCM disease relies mainly on direct observation of the fungus. However, culture growing is slow and, too often, definite diagnosis can only be obtained at later growing stages. Immunodiagnosis is also widely employed, although usually cumbersome and complex. Enzyme-based immunoassays are more amenable to automation for high-throughput testing, but may lead to cross-reactivity with other fungi. Plus, molecular diagnosis relying on polymerase-chain reaction (PCR) and nucleic-acid hybridization, although still at early stages of application to routine diagnosis of P. brasiliensis, has triggered the development of techniques for its improved specific detection, thus contributing for epidemiological studies as well. In the future, microarrays and newer biosensing technologies, coupled to new bionanotechnological tools, will certainly improve diagnosis of PCM and other mycosis through very specific and sensitive pathogen biomolecular detection.     

Label-free genotyping of single-nucleotide polymorphisms for DNA and RNA targets using a cationic polythiophene derivate

By using the specific primer extension reaction, a new assay for genotyping of single-nucleotide polymorphisms (SNPs) has been demonstrated. The assay relies on the conformational and colorimetric change of water-soluble polythiophene derivative, poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT), upon forming interpolyelectrolyte complex with extended double strand DNA and non-extended single strand DNA. All three kinds of SNP genotypes can be colorimetrically identified with one primer extension reaction in homogeneous solution. Moreover, combining with the specific digestion of RNA strands in the RNA/DNA hybrids, the proposed assay can also be applied to SNP genotyping for RNA templates. The SNP genotyping assay does not require chemical modification of oligonucleotide probes and nucleic acid targets and any separation step. It would be useful for routinely SNP detection in ordinary laboratories.     

A comprehensive in Silico analysis of the functional and structural impact of single nucleotide polymorphisms (SNPs) in the human IL-33 gene

Interleukin 33 (IL-33) is the latest member of the IL-1 cytokine family, which plays both pro - and anti-inflammatory functions. Numerous Single-nucleotide polymorphisms (SNPs) in the IL-33 gene have been recognized to be associated with a vast variety of inflammatory disorders. SNPs associated studies have become a crucial approach in uncovering the genetic background of human diseases. However, distinguishing the functional SNPs in a disease-related gene from a pool of both functional and neutral SNPs is a major challenge and needs multiple experiments of hundreds or thousands of SNPs in candidate genes. This study aimed to identify the possible deleterious SNPs in the IL-33 gene using bioinformatics predictive tools. The nonsynonymous SNPs (nsSNPs) were analyzed by SIFT, PolyPhen, PROVEAN, SNP&GO, MutPred, SNAP, PhD SNP, and I-Mutant tools. The Non-coding SNPs (ncSNPs) were also analyzed by SNPinfo and RegulomeDB tools. In conclusion, our in-silico analysis predicted 5 nsSNPs and 22 ncSNPs as potential candidates in the IL-33 gene for future genetic association studies.     

Genotyping of single nucleotide polymorphisms by primer extension reaction and a dual-analyte bio/chemiluminometric assay

Primer extension reaction (PEXT) is the most widely used approach to genotyping of single nucleotide polymorphisms (SNP). It is based on the high accuracy of nucleotide incorporation by the DNA polymerase. We propose a dual-analyte bio/chemiluminometric method for the simultaneous detection of the PEXT reaction products of the normal and mutant allele in a high sample-throughput format. PCR-amplified DNA fragments that span the SNP of interest are subjected to two PEXT reactions using normal and mutant primers in the presence of digoxigenin-dUTP and biotin-dUTP. Both primers contain a d(A)₃₀ segment at the 5′-end but differ in the final nucleotide at the 3′-end. Under optimized conditions only the primer that is perfectly complementary with the interrogated DNA will be extended by DNA polymerase and lead to a digoxigenin- or biotin-labeled product. The products of the PEXT reactions are mixed, denatured, and captured in microtiter wells through hybridization with immobilized oligo(dT) strands. Detection is performed by adding a mixture of antidigoxigenin–alkaline phosphatase (ALP) conjugate and a streptavidin–aequorin conjugate. The flash-type bioluminescent reaction of aequorin is triggered by the addition of Ca²⁺. ALP is then measured by adding the appropriate chemiluminogenic substrate. The method was evaluated by genotyping two SNPs of the human mannose-binding lectin gene (MBL2) and one SNP of the cytochrome P450 gene CYP2D6. Patient genotypes showed 100% concordance with direct DNA sequencing data.     

The Renin-Angiotensin System: A Key Role in SARS-CoV-2-Induced COVID-19

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), was first identified in Eastern Asia (Wuhan, China) in December 2019. The virus then spread to Europe and across all continents where it has led to higher mortality and morbidity, and was declared as a pandemic by the World Health Organization (WHO) in March 2020. Recently, different vaccines have been produced and seem to be more or less effective in protecting from COVID-19. The renin–angiotensin system (RAS), an essential enzymatic cascade involved in maintaining blood pressure and electrolyte balance, is involved in the pathogenicity of COVID-19, since the angiotensin-converting enzyme II (ACE2) acts as the cellular receptor for SARS-CoV-2 in many human tissues and organs. In fact, the viral entrance promotes a downregulation of ACE2 followed by RAS balance dysregulation and an overactivation of the angiotensin II (Ang II)–angiotensin II type I receptor (AT1R) axis, which is characterized by a strong vasoconstriction and the induction of the profibrotic, proapoptotic and proinflammatory signalizations in the lungs and other organs. This mechanism features a massive cytokine storm, hypercoagulation, an acute respiratory distress syndrome (ARDS) and subsequent multiple organ damage. While all individuals are vulnerable to SARS-CoV-2, the disease outcome and severity differ among people and countries and depend on a dual interaction between the virus and the affected host. Many studies have already pointed out the importance of host genetic polymorphisms (especially in the RAS) as well as other related factors such age, gender, lifestyle and habits and underlying pathologies or comorbidities (diabetes and cardiovascular diseases) that could render individuals at higher risk of infection and pathogenicity. In this review, we explore the correlation between all these risk factors as well as how and why they could account for severe post-COVID-19 complications.