Method For Single Nucleotide Polymorphisms Detection Research Articles

Benzimidazole Resistance-Associated Mutations in the β-tubulin Gene of Hookworms: A Systematic Review.

There is a growing number of reports on the occurrence of benzimidazole resistance-associated single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various helminths of veterinary, and public health concerns. However, a comprehensive analysis of their occurrence, and their contributions to conferring benzimidazole resistance among hookworms has yet to be done. The objectives of this systematic review are to summarize and synthesize peer-reviewed evidence on the occurrence of these resistance-associated mutations in hookworms, document their geographical distribution, and assess their contributions to conferring phenotypic resistance. Three databases were systematically searched using specific keywords. Research that assessed the occurrence of benzimidazole resistance-associated SNPs in hookworms, papers that reported the geographical distribution of these SNPs, and studies that investigated the SNPs' resistance-associated phenotypic effects were included in the review. Research that was not done in hookworms, papers not in the English language, and literature reviews and book chapters were excluded. Critical appraisal checklists were used to determine the risk of bias in the selected papers. Data were extracted from the selected studies and analyzed. PROSPERO Systematic Review Protocol Registration No.: CRD42024510924. A total of 29 studies were included and analyzed. Of these, four were conducted in a laboratory setting, eight described the development and validation of SNP detection methods, and the remaining 17 involved field research. Seven SNP-induced amino acid substitutions at four loci were reported among several hookworm species: Q134H, F167Y, E198A, E198K, E198V, F200Y, and F200L. SNPs have been reported in isolates occurring in the United States, Canada, Brazil, Haiti, Australia, New Zealand, Kenya, Ghana, Mozambique, and Tanzania. Resistance mutations have not been reported in Asia. E198A and F200L were reported in Ancylostoma ceylanicum with laboratory-induced resistance. F167Y and Q134H conferred resistance in A. caninum, as revealed by in vitro investigations and field assessments. There is insufficient peer-reviewed evidence to prove the association between SNP occurrence and resistance. Mutations in the β-tubulin isotype 1 gene confer benzimidazole resistance in A. caninum and A. ceylanicum, but similar evidence is lacking for other human hookworms. Understanding benzimidazole resistance through further research can better inform treatment, prevention, and control strategies.

Optimization and Clinical Application Potential of Single Nucleotide Polymorphism Detection Method Based on CRISPR/Cas12a and Recombinase Polymerase Amplification.

Conventional methods for detecting single nucleotide polymorphisms (SNPs) in clinical practice often require substantial time, labor, and specialized equipment, limiting their widespread application. To address this limitation, we refined our previous SNP detection method, IMAS-RPA [introducing an extra mismatched base adjacent to the single-base mutant site by recombinase polymerase amplification (RPA)], resulting in an updated version termed IMAS-RPAv2. We began by introducing a suboptimal protospacer adjacent motif (PAM) sequence, GTTG, into the double-stranded DNA (dsDNA) products using either RPA or reverse transcription RPA. This modification decreased the efficiency with which CRISPR RNA (crRNA) recognizes the PAM and locally unwinds the dsDNA to form an R loop. After a delay, the R loop forms. However, due to the intentional incorporation of a mismatched base on the crRNA relative to the wild-type double-stranded DNA (WT-dsDNA), a continuous two-base mismatch is established between the crRNA and WT-dsDNA. Consequently, WT-dsDNA does not activate CRISPR/Cas12a's cleavage activity within a short time, while variant-type dsDNA continues to activate CRISPR/Cas12a and produce a robust fluorescence signal. This improvement significantly enhances the SNP discrimination sensitivity, allowing for detection at the single-copy level. Results were observed using both a conventional microplate reader and a specially designed portable device created through 3D printing. This device allows a direct fluorescence observation without the need for additional equipment. Consequently, the entire detection process becomes independent of large-scale equipment. This greatly expands its range of applications and offers promising prospects for clinical use.

A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations

Single-nucleotide polymorphism (SNP) detection is critical for diagnosing diseases, and the development of rapid and accurate diagnostic tools is essential for treatment and prevention. Allele-specific polymerase chain reaction (AS-PCR) is widely used for detecting SNPs with multiplexing capabilities, while CRISPR-based technologies provide high sensitivity and specificity in targeting mutation sites through specific guide RNAs (gRNAs).In this study, we have integrated the high sensitivity and specificity of CRISPR technology with the multiplexing capabilities of AS-PCR, achieving the simultaneous detection of ten single-base mutations. As for Multi-AS-PCR, our research identified that competitive inhibition of primers targeting the same loci, coupled with divergent amplification efficiencies of these primers, could result in diminished amplification efficiency. Consequently, we adjusted and optimized primer combinations and ratios to enhance the amplification efficacy of Multi-AS-PCR. Finally, we successfully developed a novel nested Multi-AS-PCR-Cas12a method for multiplex SNPs detection. To evaluate the clinical utility of this method in a real-world setting, we applied it to diagnose rifampicin-resistant tuberculosis (TB). The limit of detection (LoD) for the nested Multi-AS-PCR-Cas12a was 102 aM, achieving sensitivity, specificity, positive predictive value, and negative predictive value of 100 %, 93.33 %, 90.00 %, and 100 %, respectively, compared to sequencing.In summary, by employing an innovative design that incorporates a universal reverse primer alongside ten distinct forward allele-specific primers, the nested Multi-AS-PCR-Cas12a technique facilitates the parallel detection of ten rpoB gene SNPs. This method also holds broad potential for the detection of drug-resistant gene mutations in infectious diseases and tumors, as well as for the screening of specific genetic disorders.

A novel AllGlo probe-quantitative PCR method for detecting single nucleotide polymorphism in CYP2C19 to evaluate the antiplatelet activity of clopidogrel

CYP2C19 gene has multiple single nucleotide polymorphism (SNP), which is the major determinant for clopidogrel treatment responses. Therefore, CYP2C19 SNP detection is essential for predicting clopidogrel efficacy. Currently, there is still no quick and effective method for routine detection of common CYP2C19 SNPs in clinical laboratories, which is critically needed prior to clopidogrel treatment. AllGlo™ based quantitative PCR was used to develop a novel genotyping method for CYP2C19 SNP detection, termed CyPAllGlo. The performance of CyPAllGlo was compared with that of the commonly used fluorescence in situ hybridization (FISH) method, and the data was verified by DNA sequencing. CyPallGlo was used to identify CYP2C19 polymorphisms in 363 patients with coronary heart disease. The univariate analysis was used to access the antiplatelet efficacy of clopidogrel in patients. The associations between CYP2C19 polymorphisms and clopidogrel efficacy were analyzed. Using CyPAllGlo to detect CYP2C19*2 and CYP2C19*3 alleles was highly specific and fast. The detection limit was approximately 0.07 µg/µl and 0.7 µg/µl for CYP2C19*2 and CYP2C19*3, respectively. The consistency between FISH and CyPAllGlo were 98.07% for CYP2C19*2 and 99.17% for CYP2C19*3. DNA sequencing showed that the accuracy of CyPAllGlo was 100%. The analysis time for the whole CyPAllGlo procedure was approximately 60 min. Univariate analysis showed that the anticoagulation efficacy of clopidogrel was related to patient age, CYP2C19 genotype, metabolic phenotype, and LDL level. The logistic regression analysis showed that the genotype of CYP2C19 and metabolic phenotype was the two risk factors for clopidogrel antiplatelet ineffectiveness. This novel CyPAllGlo is a rapid and accurate method for detection of CYP2C19 SNP. The specificity and consistency of CyPAllGlo are comparable with that of widely used DNA sequencing. These findings provide valuable rapid method for predicting clopidogrel efficacy, which can be quickly translated to improve personalized precision medicine for coronary heart disease treatment.

The Development of Multiplex PCR-RFLP of FTO Genetics Variants

Background: Major health concern throughout the whole world is the increasing rate of obesity, either in elder group or younger group of multiracial ethnics that are believed to be associated with genetic factor that plays important role in the pathogenesis of obesity. The very first gene to be associated with obesity is fat mass and obesity-associated (FTO) gene with the involvement from many single nucleotide polymorphisms (SNPs) in the first intron of FTO. Early detection of obesity-related SNPs is very crucial in the effort of reducing obesity rate which is expected to increase by 2025.This has led to the urge of developing a method to detect individual with predisposing factors towards obesity, with the hope that this method will eventually helpful in assisting healthcare authorities to rapidly detect individual who are at high risk of being obese. Multiplex PCR-RFLP is a method for rapid detection of obesity-related SNPs using gene sequence amplifying technique and optimization done using gradient PCR technique. Thus, this study was conducted to evaluate the potential of multiplex PCR-RFLP to amplify FTO genetic variants. Materials and methods: A total of 14 UniSZA students were recruited to obtain 2 ml of peripheral whole blood for DNA extraction. Single gradient PCR dan multiplex PCR were employed to determine the optimum annealing temperature for all assigned FTO SNPs as rs9939609, rs1421085 and rs17817288. The PCR products were run on 1% of agarose gel electrophoresis and visualized under UV Gel doc Image Analyzer. These PCR products were introduced to restriction enzymes for the cutting site on genetic variation namely Bfal, HpyCH4V, HpyCH4IV for rs9939609, rs1421085 and rs17817288, respectively. Next, the PCR fragments were identified on 2% agarose gel electrophoresis under UV Gel Doc Image Analyzer. Results: The optimum annealing temperature to amplify all FTO SNPs obtained through gradient PCR was 60°C. The amplification of FTO rs9939609, rs17817288 and rs1421085 regions, showed specific size product of 151 bp, 140 bp, and 131 bp respectively. In RFLP, the fragments after restriction enzyme digestion produced homozygous wild type, heterozygous and homozygous mutant type for genotype characterization based on allele cut site by specific restriction enzyme namely BfaI, HpyCG4IV and HpyCH4V, respectively. Conclusion: This study may become preliminary data on the potential of multiplex PCR-RFLP in provide rapid diagnosis to amplify multiple FTO genetic variants.

A FEN 1-driven DNA walker-like reaction coupling with magnetic bead-based separation for specific SNP detection.

Single-nucleotide polymorphism (SNP) plays a key role in the carcinogenesis of the human genome, and understanding the intrinsic relationship between individual genetic variations and carcinogenesis lies heavily in the establishment of a precise and sensitive SNP detection platform. Given this, a powerful and reliable SNP detection platform is proposed by a flap endonuclease 1 (FEN 1)-driven DNA walker-like reaction coupling with a magnetic bead (MB)-based separation. A carboxyfluorescein (FAM)-labeled downstream probe (DP) was decorated on a streptavidin magnetic bead (SMB). The target DNA, as a walker strand, was captured by hybridization with DP and an upstream probe (UP) to form a three-base overlapping structure and execute the walking function on the surface of SMB. FEN 1 was employed to specifically recognize the three-base overlapping structure and cut the 5'flap at the SNP site to report the walking event and signal amplification. Considering the fact that the fluorescence was labeled on the cleavage and uncleavage sequences of DP and the target DNA-triggered walking event was undistinguishable from the mixtures, magnetic separation came in handy for cleavage probe (CP) isolation and discrimination of the amplified signal from the background signal. In comparison with the conventional DNA walker reaction, this strategy was coupling with SMB-based separation, thus promising a powerful and reliable method for SNP detection and signal amplification.

Modified Allele-Specific qPCR (ASQ) Genotyping.

The allele-specific qPCR (ASQ) method for SNP (single nucleotide polymorphism) detection is based on the FRET (fluorescence resonance energy transfer) system, a system using position-dependent fluorescent dyes and quenches. The modified ASQ method requires two separate components: (1) the allele-specific part, two AS primers targeting the SNP with identity in the penultimate positions atthe 3'-end and specific tags in the 5'-end, and (2) the universal part, two universal probes (UPs) with corresponding tags and different fluorescent dyes in the 5'-end and a single common universal probe with a quencher in the 3'-ends (Uni-Q), complementary to all UP tags. There are two major variations of the ASQ method, with either short 4-bp tags (variant A) or longer 6-bp tags (variant B), both of which have been successfully used for SNP genotyping in plants. The modified ASQ method is much cheaper compared to other similar FRET-based methods because the most expensive parts, the universal probes, have a short and linear structure, where fluorophores and quenchers are located in the ends but not incorporated inside of the sequences.

CASMART, a one-step CRISPR Cas12a-mediated isothermal amplification for rapid and high-resolution digital detection of rare mutant alleles

Convenient, ultrasensitive, and accurate detection of rare variants is essential for early cancer diagnosis and precision medicine, however, despite years of efforts, tools that have all these qualities remain elusive. Here, we developed a one-step CRISPR/Cas12a-based digital diagnostic platform for accurately quantifying mutant alleles, referred to as the CRISPR ASsoaciated Mutation Allele Rapid Test (CASMART). The platform accurately quantifies the variant allele frequency of EGFR L858R within 1 h at 42 °C and can detect mutant targets as low as 0.3 copies/μL (0.498 aM) in mock multiplex cfDNA samples. We further investigated the applicability of CASMART using human genomic samples with confirmed EGFR L858R mutations previously measured variant allele frequency by next-generation sequencing. Comparison across platforms revealed equivalent detection performance (Pearson's correlation coefficient, R2 = 0.9208) and high quantification accuracy for mutation allele frequency (intraclass correlation coefficient = 0.959). Our one-step approach enables easy and accurate variant allele frequency measurement of rare mutant alleles without PCR instrumentation, while the assay time was reduced by approximately half compared to the digital PCR with the shortest turnaround. The CASMART is an alternative to conventional single nucleotide polymorphism detection methods with great potential as a next-generation biosensor for rapidly quantifying the variant allele fraction, especially in resource-limited settings.

The Role of Single-Nucleotide Polymorphisms in Cholangiocarcinoma: A Systematic Review

Single-nucleotide polymorphisms (SNPs) play an essential role in various malignancies, but their role in cholangiocarcinoma (CCA) remains to be elucidated. Therefore, the purpose of this systematic review was to evaluate the association between SNPs and CCA, focusing on tumorigenesis and prognosis. A systematic literature search was carried out using PubMed, Embase, Web of Science and the Cochrane database for the association between SNPs and CCA, including literature published between January 2000 and April 2022. This systematic review compiles 43 SNPs in 32 genes associated with CCA risk, metastatic progression and overall prognosis based on 34 studies. Susceptibility to CCA was associated with SNPs in genes related to inflammation (PTGS2/COX2, IL6, IFNG/IFN-γ, TNF/TNF-α), DNA repair (ERCC1, MTHFR, MUTYH, XRCC1, OGG1), detoxification (NAT1, NAT2 and ABCC2), enzymes (SERPINA1, GSTO1, APOBEC3A, APOBEC3B), RNA (HOTAIR) and membrane-based proteins (EGFR, GAB1, KLRK1/NKG2D). Overall oncological prognosis was also related to SNPs in eight genes (GNB3, NFE2L2/NRF2, GALNT14, EGFR, XRCC1, EZH2, GNAS, CXCR1). Our findings indicate that multiple SNPs play different roles at various stages of CCA and might serve as biomarkers guiding treatment and allowing oncological risk assessment. Considering the differences in SNP detection methods, patient ethnicity and corresponding environmental factors, more large-scale multicentric investigations are needed to fully determine the potential of SNP analysis for CCA susceptibility prediction and prognostication.

Universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR) for SNP genotyping

BackgroundThe detection and identification of single nucleotide polymorphism (SNP) is essential for determining patient disease susceptibility and the delivery of medicines targeted to the individual. At present, SNP genotyping technology includes Sanger sequencing, TaqMan-probe quantitative polymerase chain reaction (qPCR), amplification-refractory mutation system (ARMS)-PCR, and Kompetitive Allele-Specific PCR (KASP). However, these technologies have some disadvantages: the high cost of development and detection, long and time consuming protocols, and high false positive rates. Focusing on these limitations, we proposed a new SNP detection method named universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR). In this method, only two types of fluorescence-labeled probes were used for SNP genotyping, thus greatly reducing the cost of development and detection for SNP genotyping.ResultsIn the amplification process of UPIP-qPCR, unlabeled intermediate primers with template-specific recognition functions could trigger probe hydrolysis and specific signal release. UPIP-qPCR can be used successfully and widely for SNP genotyping. The sensitivity of UPIP-qPCR in SNP genotyping was 0.01 ng, the call rate was more than 99.1%, and the accuracy was more than 99.9%. High-throughput DNA microarrays based on intermediate primers can be used for SNP genotyping.ConclusionThis novel approach is both cost effective and highly accurate; it is a reliable SNP genotyping method that would serve the needs of the clinician in the provision of targeted medicine.

Highly selective detection of single nucleotide polymorphism (SNP) using a dumbbell DNA probe with a gap-filling approach

Single nucleotide polymorphism (SNP) is the most common type of genetic sequence variations and known to associate with the development of various diseases including cancer. Accurate detection of SNP is often necessary to determine the susceptibility of patients to diseases as well as their response to the medical treatments. Molecular inversion probe (MIP) has been widely utilized as an alternative method to detect the SNP as compared to the conventional PCR/genome sequencing method. However, base-pairing based detection of SNP fundamentally lacks target specificity toward the single mismatch of bases and often fails to meet the accuracy requirement for SNP detection. In this study, we report the development of new SNP detection method using a dumbbell-probe with a gap-filling approach. Two separate DNA strands are designed to self-assemble to form a dumbbell DNA probe for the selective detection of EGFR 21 point mutation. Unlike the direct binding of MIP to the target sequence, our system has an additional process for the enhancement of sequence specificity through a base matched gap-filling. In the presence of target sequences, the dumbbell-probe undergoes base-pair hybridization to the target sequence followed by sequence specific gap-filling and ligation in single-step. The detection of EGFR 21 point mutation is achieved with a highly specific and selective manner. This simple and robust detection method offers promising future potential in the diagnosis of various genetic mutations.

Development of two different pharmacogenomics detection methods for second-generation antipsychotic drugs quetiapine and aripiprazole

Abstract Objective: Second-generation antipsychotics are widely used in mental illness, but the treatment effects and side effects are affected by single nucleotide polymorphisms (SNPs) of related genes. Quetiapine and aripiprazole are two frequently used second-generation antipsychotic drugs. The aim of this study was to develop two different SNP detection methods for four SNP alleles associated with the pharmacokinetics of quetiapine and aripiprazole, based on high-resolution melting (HRM) and multicolor melting curve assay (MMCA) respectively. Methods: Whole genome DNA samples were obtained from 240 healthy people (107 females and 133 males) without genetic diseases. HRM methods were established using four kinds of specific primers and a saturated fluorescent dye. Each SNP allele with their own primers was detected in a single reaction. In the MMCA method, a multiplex polymerase chain reaction with 4 different-colored fluorescent probes was established to detect four SNP alleles in a single reaction. All experimental protocols were approved by the Ethics Committee of the Shanghai Children's Medical Center, China (SCMC-201015) on November 22, 2010. Results: Two detection methods for the pharmacogenomics of quetiapine and aripiprazole, based on HRM and MMCA respectively, were established in this study. The single-target HRM method can be completed in 96 minutes, whereas the quadruplex MMCA method takes 133 minutes. It was found that the results of HRM and MMCA for the four SNP alleles had 100% coincidence with Sanger sequencing in the 240 samples. Conclusion: This study developed two methods for the detection of four pharmacogenomic SNP alleles that correlated with quetiapine and aripiprazole. Both methods are rapid, cost-saving, highly accurate and potentially facilitate rational use of second-generation antipsychotics for clinical medication.

A Microfluidic-Based SNP Genotyping Method for Hereditary Hearing-Loss Detection.

The genotyping of SNPs (single nucleotide polymorphisms) is a prerequisite for the analysis of many genetic diseases, including hereditary hearing-loss. However, the existing methods for SNP detection suffer from a long detection period, tedious operation, and a high risk of carryover contamination. To address these challenges, a microfluidic chip is constructed for rapid and efficient SNP genotyping by dividing the sample into many independent chambers for Kompetitive Allele Specific PCR in this study. Using this strategy, multiple detection can be easily accomplished and the challenge for the establishment of multiplex PCR is fundamentally overcome. The entire detection can be finished within 2 h in a fully sealed manner with this method, which is quite simple compared to SNaPshot and MassArray. After assessment of the basic performance, this chip was applied to screen 15 mutations, including SNPs and InDels (insertion-deletion markers), that can cover more than 80% of cases of hereditary hearing-loss in China. Over 40 clinical samples were analyzed with this microfluidic chip for SNP genotyping, and the results are consistent with that obtained by Sanger sequencing, demonstrating its practicability and potential in the application of genetic disease detection.

A PCR-lateral flow assay system based on gold magnetic nanoparticles for CYP2C19 genotyping and its clinical applications

Presently, clopidogrel is the standard therapeutic drugs for antiplatelet therapy. Variants in the CYP2C19 gene influence the clinical response of clopidogrel. Thus, the US Food and Drug Administration suggested CYP2C19 genotyping needs to identify before taking medicine. Due to high cost, time consuming, and sophisticated instruments, current single nucleotide polymorphism detection methods are limited in clinical application. In the present study, we established a genotyping method for CYP2C19, which combines amplification refractory mutation system (ARMS)-PCR with a lateral flow assay used gold magnetic nanoparticles (GMNPs) named as PCR-gold magnetic lateral flow assay system (PCR-GoldMag LFA). The PCR products with specific genotype can be explained within 5 minutes, either through visual or by a magnetic reader automatically according to the captured GMNPs probes on the test lines of the LFA strips. The limit of detection of this method is 5 ng of genomic DNA. The PCR-GoldMag LFA system was applied in a clinical trial with 1356 samples of Han Chinese. The concordance rate between the LFA system and sequencing is 99.93%. The allele frequency of CYP2C19*2 and CYP2C19*3 are 30.38 and 7.08% in Han Chinese, respectively. This method provides a new way in the clinical application of CYP2C19 genotyping to guide the clopidogrel medication.

A method for SNP detection using MoS2@AuNPs and SYBR Green I in combination with enzyme digestion

Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases.

SNP-Based Kompetitive Allele Specific PCR (KASPTM) Method for the Qualification and Quantification of Hop Varieties

There are several studies focused on single nucleotide polymorphisms (SNPs) of hops. However, to date, no feasible method for the qualification and quantification of hop varieties had been developed. A novel Kompetitive Allele Specific PCR (KASP) methodology for the qualification and quantification of hop varieties has been developed. One hundred and fifty gauge of high-throughput sequencing data were obtained from the ten hop varieties most commonly used in the Chinese brewing industry. After bioinformatic analysis, 2046 SNPs from sixteen hop varieties were selected from two million original SNPs for further confirmation by Sanger sequencing. Twelve SNP markers were successfully screened for the characterization and quantification of the mixed sample. The SNP markers and detection method developed provide potential means for the qualification and quantification of hop varieties in laboratories utilizing SNP marker technology.

An application of competitive reporter monitored amplification (CMA) for rapid detection of single nucleotide polymorphisms (SNPs).

Single nucleotide polymorphisms (SNPs) are essential parameters in molecular diagnostics and can be used for the early detection and clinical prognosis in various diseases. Available methods for SNP detection are still labor-intensive and require a complex laboratory infrastructure, which are not suitable for the usage in resource-limited settings. Thus, there is an urgent need for a simple, reliable and rapid approach. In this paper we modified the previously developed competitive reporter monitored amplification (CMA) technique for the detection of resistance mediating SNPs in Mycobacterium tuberculosis complex (MTBC) strains. As a proof-of-principle for the application of the CMA-based SNP assay in routine molecular tuberculosis diagnostic, we show that the assay recognizes resistance mediating SNPs for rifampicin, isoniazid and ethambutol from either isolated DNA or heat inactivated M. tuberculosis cell cultures. The CMA-based SNP assay can identify the most prevalent resistance mediating mutations in the genes rpoB, katG, embB, and the promotor region of inhA within one hour.

Ru(bpy)32+ Incorporated Luminescent Polymer Dots: Double-Enhanced Electrochemiluminescence for Detection of Single-Nucleotide Polymorphism.

Encapsulating a large amount of electrochemiluminescence (ECL) active molecules into one nanoparticle for single-target recognition can greatly improve the sensitivity of the ECL assay. This work used luminescent conjugated polymer as a carrier to synthesize Ru(bpy)32+-doped polymer dots (RuPdots). The RuPdots with an average size of 20 nm and encapsulation of about 1354 Ru(bpy)32+ showed greatly enhanced ECL emission. Using tripropylamine as a coreactant, the resonance energy transfer from the excited polymer dots to the encapsulated Ru(bpy)32+ was demonstrated, which led to a 15.7-fold higher ECL emission than that of Ru(bpy)32+ with the same amount at the gold electrode. By coupling the double-enhanced ECL signal of this nanostructure with the specific ligase detection reaction, a sensitive ECL biosensor was constructed for specific detection of a single-nucleotide polymorphism (SNP). Using mutant KRAS gene as a model target, the ECL SNP detection method showed a linear range from 1 fM to 1 nM and a detection limit of 0.8 fM. The designed RuPdots provided a novel signal amplification strategy and could be used as a universal label for ultrasensitive ECL biodetection.

Development of methods for detection of SNPs in Activin type IIB receptor and a preliminary study on association between its polymorphism and growth parameters of the Asian seabass Lates calcarifer

Molecular markers that allow selection of juveniles and broodstock with a high growth performance are useful for the aquaculture industry. In this study, association between single nucleotide polymorphisms (SNPs) of the Activin type IIB receptor (LcActRIIB) and growth-related parameters of Asian seabass Lates calcarifer juveniles were examined by PCR-direct sequencing. Three SNPs (G>Ac.965+g.7, C>A>T c.965+g.33 and G>A c.965+g.189) generating seven composite SNP genotypes (diplotypes) were found in the LcActRIIB gene segment (398 bp). Among three identified SNPs, only a G>Ac.965+g.7 SNP showed significant association with growth traits (average body weight, total length, hepatic weight and hepatosomatic index) of examined fish (P Ac.965+g.7 SNP were successfully developed based on gel-based bi-allelic PCR amplification of specific alleles (Bi-PASA) and real-time PCR-based PASA analyses (N = 99). Identical genotypes were consistently obtained from different detection methods.

SNP-E: A New Method For Multiple Sequence Alignments Analysis And Accurate Single Nucleotide Polymorphism Evaluation

Identification of single nucleotide polymorphisms (SNPs) and insertion-deletion mutations are important for discovering the connection between the genetic mutations and complex diseases. The objective of this study was to develop a sensitive and accurate computational method for SNP detection among Multiple Sequence Alignments (MSAs) to be run on Microsoft Office SuiteTM and WindowsTM. The SNP-Evaluator, was designed to simulate the process of human eye visual change-identification. Analysis of three 82-Kbp genomic loci derived from Sanger sequencing and the corresponding SNPs from 31 genomes from IlluminaTM sequencing of soybean (Glycine max L. Merr.) demonstrated that the SNP-E was an effective method for medium-scale genomic research.