Detection of single nucleotide polymorphism by measuring extension kinetics with T7 exonuclease mediated isothermal amplification

The Analyst ◽  
2018 ◽  
Vol 143 (1) ◽  
pp. 116-122 ◽  
Author(s):  
Miao Cui ◽  
Xianjin Xiao ◽  
Meiping Zhao ◽  
Bo Zheng
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Sensitivity And Specificity ◽  
Room Temperature ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
T7 Exonuclease

Kinetics based detection of single nucleotide polymorphism at room temperature with high sensitivity and specificity.

scholarly journals A Novel Single-Nucleotide Polymorphism Loop Mediated Isothermal Amplification Assay for Detection of Artemisinin-Resistant Plasmodium falciparum Malaria

2018 ◽  
Vol 5 (4) ◽  
Author(s):  
Abu Naser Mohon ◽  
Didier Menard ◽  
Mohammad Shafiul Alam ◽  
Kevin Perera ◽  
Dylan R Pillai
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Dynamic Range ◽  
Developed Countries ◽  
Isothermal Amplification ◽  
Test Method ◽  
Nucleotide Polymorphism ◽  
In Vitro Susceptibility ◽  
Single Nucleotide ◽  
Loop Mediated Isothermal Amplification

Abstract Background Artemisinin-resistant malaria (ARM) remains a significant threat to malaria elimination. In the Greater Mekong subregion, the prevalence of ARM in certain regions has reached greater than 90%. Artemisinin-resistant malaria is clinically identified by delayed parasite clearance and has been associated with mutations in the propeller domain of the kelch 13 gene. C580Y is the most prevalent mutation. The detection of ARM currently relies on labor-intensive and time-consuming methods such as clinical phenotyping or in vitro susceptibility testing. Methods We developed a novel single-nucleotide polymorphism loop mediated isothermal amplification (SNP-LAMP) test method for the detection of the C580Y mutation using a novel primer design strategy. Results The SNP-LAMP was 90.0% sensitive (95% confidence interval [CI], 66.9–98.3) and 91.9% specific (95% CI, 82.6–96.7) without knowledge of the parasite load and was 100% sensitive (95% CI, 79.9–100) and 97.3% specific (95% CI, 89.7–99.5) when the parasitemia was within the assay dynamic range. Tests with potential application near-to-patient such as SNP-LAMP may be deployed in low- and middle-income and developed countries. Conclusions Single-nucleotide polymorphism LAMP can serve as a surveillance tool and guide treatment algorithms for ARM in a clinically relevant time frame, prevent unnecessary use of additional drugs that may drive additional resistance, and avoid longer treatment regimens that cause toxicity for the patient.

193 BOVINE EMBRYO GENOTYPING USING A 50K SINGLE NUCLEOTIDE POLYMORPHISM CHIP

2011 ◽  
Vol 23 (1) ◽  
pp. 197 ◽  
Author(s):  
A. D. Le Bourhis ◽  
E. Mullaart ◽  
P. Humblot ◽  
W. Coppieters ◽  
C. Ponsart
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Room Temperature ◽  
Drop Out ◽  
Bovine Embryo ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Good Efficiency ◽  
Student’S T

Genomic tools are now available for most livestock species and are used routinely for marker-assisted selection (MAS) and genomic selection (GS) in cattle. Recently, multiple-marker detection has been achieved from biopsies of preimplantation stage embryos, thus allowing embryos to be selected before transfer (Le Bourhis et al. 2009 Reprod. Fertil. Dev. 21, 192 abst). This strategy provides the opportunity to estimate some traits of particular interest, the presence of genetic abnormalities, or both. The present work aimed to assess the efficiency of MAS/GS evaluation from biopsied bovine embryos by using the bovine 50K single nucleotide polymorphism (SNP) Illumina chip. A biopsy of 5 to 10 cells was obtained under laboratory conditions, using a microblade under a stereomicroscope, from 29 in vitro-cultured morulae and blastocysts. Biopsies were transferred individually as dry samples in tubes and sent frozen (n = 13) or at room temperature (n = 16) to the genotyping laboratory. The genomic DNA of each biopsy was amplified using a whole-genome amplification (WGA) kit according to the manufacturer’s instructions (Qiagen REPLI-g® Mini Kit, Qiagen, Valencia, CA). Following WGA, DNA concentration was determined by using PicoGreen. For subsequent genotyping, a custom CRV 50K Illumina chip was used. Call rates were calculated from 50 905 SNP. Percentage of allele drop-out (%ADO), which was estimated from the number of heterozygous markers [%ADO = (calculated hetero – observed hetero)/calculated hetero]. Parentage error was estimated from 12 embryos by using the genotypes of the parents of the embryos. Both groups of transport conditions were compared using Student’s t-test. Results are presented as mean ± SEM. A greater quantity of DNA was obtained after amplification of biopsies that were sent frozen to the laboratory when compared with those at room temperature (P < 0.05). However, the SNP call rate, %ADO, and parentage error did not differ between groups. These results indicate that genotyping from embryo biopsies following WGA can be achieved with good efficiency when using high-density marker chips. To validate the use of MAS/GS from early embryos in breeding schemes, a larger number of in vivo embryos are currently genotyped under field conditions. This will allow the reliability of this method to be assessed and the correlation between embryo and calf genetic evaluation to be quantified with the current WGA efficiency. Table 1.Amount of DNA after WGA and genotyping results

scholarly journals Simultaneous multiple single nucleotide polymorphism detection based on click chemistry combined with DNA-encoded probes

2018 ◽  
Vol 9 (13) ◽  
pp. 3335-3340 ◽  
Author(s):  
Qian-Yu Zhou ◽  
Fang Yuan ◽  
Xiao-Hui Zhang ◽  
Ying-Lin Zhou ◽  
Xin-Xiang Zhang
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Click Reaction ◽  
High Sensitivity ◽  
Dna Ligase ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide Polymorphism Detection ◽  
Single Nucleotide ◽  
Polymorphism Detection ◽  
Novel Strategy ◽  
Dna Template

A novel strategy utilizing a DNA template-directed CuAAC click reaction to mimic a ligation reaction based on DNA ligase was successfully established for multiple SNP detection with high sensitivity and specificity.

Room-Temperature Single-Nucleotide Polymorphism and Multiallele DNA Detection Using Fluorescent Nanocrystals and Microarrays

2003 ◽  
Vol 75 (18) ◽  
pp. 4766-4772 ◽  
Author(s):  
Daniele Gerion ◽  
Fanqing Chen ◽  
Balaji Kannan ◽  
Aihua Fu ◽  
Wolfgang J. Parak ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Room Temperature ◽  
Dna Detection ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

scholarly journals Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High-Resolution Melting Analysis

2004 ◽  
Vol 50 (10) ◽  
pp. 1748-1754 ◽  
Author(s):  
Gudrun H Reed ◽  
Carl T Wittwer
Keyword(s):  
Single Nucleotide Polymorphism ◽  
High Resolution ◽  
Sensitivity And Specificity ◽  
High Resolution Melting ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Product Size ◽  
Pcr Products ◽  
Melting Analysis

Abstract Background: Screening for heterozygous sequence changes in PCR products, also known as “mutation scanning”, is an important tool for genetic research and clinical applications. Conventional methods require a separation step. Methods: We evaluated the sensitivity and specificity of homogeneous scanning, using a saturating DNA dye and high-resolution melting. Heterozygous single-nucleotide polymorphism (SNP) detection was studied in three different sequence backgrounds of 40%, 50%, and 60% GC content. PCR products of 50–1000 bp were generated in the presence of LCGreen™ I. After fluorescence normalization and temperature overlay, melting curve shape was used to judge the presence or absence of heterozygotes among 1632 cases. Results: For PCR products of 300 bp or less, all 280 heterozygous and 296 wild-type cases were correctly called without error. In 672 cases between 400 and 1000 bp with the mutation centered, the sensitivity and specificity were 96.1% and 99.4%, respectively. When the sequence background and product size with the greatest error rate were used, the sensitivity of off-center SNPs (384 cases) was 95.6% with a specificity of 99.4%. Most false negatives occurred with SNPs that were compared with an A or T wild type sequence. Conclusions: High-resolution melting analysis with the dye LCGreen I identifies heterozygous single-base changes in PCR products with a sensitivity and specificity comparable or superior to nonhomogeneous techniques. The error rate of scanning depends on the PCR product size and the type of base change, but not on the position of the SNP. The technique requires only PCR reagents, the dye LCGreen I, and 1–2 min of closed-tube, post-PCR analysis.

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