High Microsatellite and SNP Genotyping Success Rates Established in a Large Number of Genomic DNA Samples Extracted From Mouth Swabs and Genotypes

2006 ◽  
Vol 9 (4) ◽  
pp. 501-506 ◽  
Author(s):  
Josine L. Min ◽  
Nico Lakenberg ◽  
Margreet Bakker-Verweij ◽  
Eka Suchiman ◽  
Dorret I. Boomsma ◽  
...  
Keyword(s):  
Success Rate ◽  
Genomic Dna ◽  
Snp Genotyping ◽  
High Yield ◽  
Success Rates ◽  
Nucleotide Polymorphism ◽  
High Quality ◽  
Single Nucleotide ◽  
Dna Yield ◽  
Yield And Quality

AbstractIn this article, we present the genomic DNA yield and the microsatellite and single nucleotide polymorphism (SNP) genotyping success rates of genomic DNA extracted from a large number of mouth swab samples. In total, the median yield and quality was determined in 714 individuals and the success rates in 378,480 genotypings of 915 individuals. The median yield of genomic DNA per mouth swab was 4.1 μg (range 0.1–42.2 μg) and was not reduced when mouth swabs were stored for at least 21 months prior to extraction. A maximum of 20 mouth swabs is collected per participant. Mouth swab samples showed in, respectively, 89% for 390 microsatellites and 99% for 24 SNPs a genotyping success rate higher than 75%. A very low success rate of genotyping (0%–10%) was obtained for 3.2% of the 915 mouth swab samples using microsatellite markers. Only 0.005% of the mouth swab samples showed a geno-typing success rate lower than 75% (range 58%–71%) using SNPs. Our results show that mouth swabs can be easily collected, stored by our conditions for months prior to DNA extraction and result in high yield and high-quality DNA appropriate for genotyping with high success rate including whole genome searches using microsatellites or SNPs.

ANALYSIS OF FOUR DIFFERENT METHODS OF DNA EXTRACTION FROM THE TRUE WEEVIL (COLEOPTERA: CURCULIONIDAE)

2018 ◽  
Vol 24 (1) ◽  
Author(s):  
DIVYA SHARMA ◽  
DALIP KUMAR ◽  
RHITOBAN RAY CHOUDHURY
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Method ◽  
Sitophilus Oryzae ◽  
Potassium Acetate ◽  
Single Specimen ◽  
High Quality ◽  
Dna Yield ◽  
Yield And Quality ◽  
Dna Extraction Method

PCR-based markers have been widely used for the analysis of genetic diversity and to avoid ambiguity, molecular characterization is very effective tool for accurate discrimination and identification of a species in insects. Because these studies require analysis of large number of samples, a DNA extraction method that is fast, inexpensive and yields high quality DNA from the preserved samples, needs to be evaluated. A comparative analysis of four methods for DNA extraction from a single specimen of rice weevil, Sitophilus oryzae preserved in 90% alcohol has been communicated. Significantly higher DNA yields were obtained by using SDS-Potassium acetate method followed by CTAB, DNA XPress and Bioline Isolate II genomic DNA kit. Maximum purity (A260/A280- 1.8) was obtained with Bioline Isolate II genomic DNA kit method. The Absorbance ratio was appreciably low with DNA Xpress kit showing the presence of proteins. Bioline Isolate II genomic DNA kit was time efficient and yielded good quality DNA but at a high cost. Based on DNA yield and quality, these evaluations provide a guide for choosing Bioline Isolate II genomic DNA kit method of DNA extraction for rice weevils and optimizing the extraction conditions for rice weevils.

scholarly journals Single Nucleotide Polymorphism Genotyping Analysis Shows That Vaccination Can Limit the Number and Diversity of Recombinant Progeny of Infectious Laryngotracheitis Viruses from the United States

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Carlos A. Loncoman ◽  
Carol A. Hartley ◽  
Mauricio J. C. Coppo ◽  
Glenn F. Browning ◽  
Gabriela Beltrán ◽  
...  
Keyword(s):  
United States ◽  
Single Nucleotide Polymorphism ◽  
Viral Replication ◽  
The United States ◽  
Snp Genotyping ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Genotyping Assay ◽  
Infectious Laryngotracheitis

ABSTRACT Infectious laryngotracheitis (ILTV; Gallid alphaherpesvirus 1) causes mild to severe respiratory disease in poultry worldwide. Recombination in this virus under natural (field) conditions was first described in 2012 and more recently has been studied under laboratory conditions. Previous studies have revealed that natural recombination is widespread in ILTV and have also demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In the United States, natural ILTV recombination has also been detected, but not as frequently as in Australia. To better understand recombination in ILTV strains originating from the United States, we developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two virulent U.S. field strains of ILTV (63140 and 1874c5) under experimental in vivo conditions. We also tested the capacity of the Innovax-ILT vaccine (a recombinant vaccine using herpesvirus of turkeys as a vector) and the Trachivax vaccine (a conventionally attenuated chicken embryo origin vaccine) to reduce recombination. The Trachivax vaccine prevented ILTV replication, and therefore recombination, in the trachea after challenge. The Innovax-ILT vaccine allowed the challenge viruses to replicate and to recombine, but at a significantly lower rate than in an unvaccinated group of birds. Our results demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination between these ILTV strains and also show that vaccination can limit the number and diversity of recombinant progeny viruses. IMPORTANCE Recombination allows alphaherpesviruses to evolve over time and become more virulent. Historically, characterization of viral vaccines in poultry have mainly focused on limiting clinical disease, rather than limiting virus replication, but such approaches can allow field viruses to persist and evolve in vaccinated populations. In this study, we vaccinated chickens with Gallid alphaherpesvirus 1 vaccines that are commercially available in the United States and then performed coinoculations with two field strains of virus to measure the ability of the vaccines to prevent field strains from replicating and recombining. We found that vaccination reduced viral replication, recombination, and diversity compared to those in unvaccinated chickens, although the extent to which this occurred differed between vaccines. We suggest that characterization of vaccines could include studies to examine the ability of vaccines to reduce viral recombination in order to limit the rise of new virulent field strains due to recombination, especially for those vaccines that are known not to prevent viral replication following challenge.

scholarly journals Prediction of sofosbuvir response using interleukin-6 serum level and single nucleotide polymorphism of interferon lambda- 4

2020 ◽  
Vol 14 (01) ◽  
pp. 80-88
Author(s):  
Amal E Saafan ◽  
Ashraf Abobaker ◽  
Mohamed S Abbas ◽  
Ahmed El-Gendy
Keyword(s):  
Serum Level ◽  
Real Time ◽  
Real Time Pcr ◽  
Dual Therapy ◽  
Snp Genotyping ◽  
Nucleotide Polymorphism ◽  
Time Intervals ◽  
Genotype 4 ◽  
Single Nucleotide ◽  
Taqman Probes

Introduction: In Egypt, 15% of the populations are suffering from chronic hepatitis C especially genotype 4. Sofosbuvir was approved by FDA in December 2013 for treatment of HCV genotypes 2 and 3 in combination with Ribavirin, and for genotypes 1 and 4 in combination with Peg-IFN. Recently, polymorphism of different genes and plasma levels of IL-6 were utilized for better prediction of HCV clearance. This study aimed at early prediction of the efficacy of HCV treatment with Sofosbuvir (Sovaldi) and comparing the antiviral efficacy of dual and triple Sovaldi combination therapy. Methodology: Blood samples were collected from 100 HCV positive patients and detected by real time PCR at three time intervals. SNP genotyping of INFL-4 gene was estimated by using real-time PCR with predesigned primers and Taqman probes. IL-6 serum level was estimated before, during and after the end of the treatment using ELISA assay based on human IL-6 KIT. Results: SNP genotyping of INFL-4 gene showed that 13.1% of patients carried ∆G/∆G, 30.4% patients had TT/TT and 56.5% patients possessed heterozygote allele ∆G/TT. Clinical data displayed that 13 patients were got relapsed at SVR 12. Serum level of IL-6 was noticed higher in HCV patients than healthy ones. Noteworthy, it was increased during treatment then decreased to a minimal level than begining of treatment. Conclusion: SNP in INFL-4 gene has displayed no effect in response to Sofosbuvir. Dual therapy had the same effect like triple therapy, so interferon could be withdrawn from the treatment regimen.

Utilizing Genomic DNA Purified From Clotted Blood Samples for Single Nucleotide Polymorphism Genotyping

2002 ◽  
Vol 126 (3) ◽  
pp. 266-270
Author(s):  
Karissa K. Adkins ◽  
Daniel A. Strom ◽  
Thomas E. Jacobson ◽  
Cara R. Seemann ◽  
Darin P. O'Brien ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Methylenetetrahydrofolate Reductase ◽  
Factor V ◽  
Single Nucleotide Polymorphism Genotyping ◽  
Nucleotide Polymorphism ◽  
High Quality ◽  
Genetic Tests ◽  
Single Nucleotide ◽  
Blood Samples ◽  
Clinical Laboratories

Abstract Context.—Linking single nucleotide polymorphisms to disease etiology is expected to result in a substantial increase in the number of genetic tests available and performed at clinical laboratories. Whole blood serves as the most common DNA source for these tests. Because the number of blood samples rises with the number of genetic tests performed, alternative DNA sources will become important. One such alternative source is clotted blood, a by-product of serum extraction. Efficiently using an already procured blood sample would limit the overall number of samples processed by clinical laboratories. Objective.—To determine if DNA purified from clotted blood can be effectively used for single nucleotide polymorphism genotyping. Design.—DNA was purified from the clotted blood of 15 donors. Single nucleotide polymorphism genotyping for the methylenetetrahydrofolate reductase and factor V Leiden mutations was performed with each DNA sample by 2 independent methods. Results.—High-quality DNA was obtained from each of the 15 individual clotted blood samples as demonstrated by UV spectrophotometric analysis, gel electrophoresis, and polymerase chain reaction amplification. The DNA was used successfully to obtain genotype data from both the methylenetetrahydrofolate reductase and factor V single nucleotide polymorphism assays for all samples tested. Conclusions.—Clotted blood is a clinically abundant sample type that can be used as a source of high-quality DNA for single nucleotide polymorphism genotyping.

scholarly journals Transcriptome and genome sequencing elucidates the molecular basis for the high yield and good quality of the hybrid rice variety Chuanyou6203

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Juansheng Ren ◽  
Fan Zhang ◽  
Fangyuan Gao ◽  
Lihua Zeng ◽  
Xianjun Lu ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Hybrid Rice ◽  
Rice Variety ◽  
Utilization Efficiency ◽  
High Yield ◽  
Mrna Editing ◽  
High Quality ◽  
Flag Leaves ◽  
Yield And Quality

AbstractThe yield heterosis of rice is sought by farmers and strong contributes to food safety, but the quality of hybrid rice may be reduced. Therefore, developing new varieties with both high yield and good quality is a heavily researched topic in hybrid rice breeding. However, the molecular mechanism governing yield heterosis and high rice quality has not been elucidated to date. In this study, a comparative transcriptomics and genomic analysis was performed on a hybrid rice variety, Chuanyou6203 (CY6203), and its parents to investigate the molecular mechanism and gene regulation network governing the formation of yield and quality stages. A total of 66,319 SNPs and InDels between CH3203 and C106B were detected in the 5′-UTR, exon, intronic, and 3′-UTR regions according to the reference genome annotation, which involved 7473 genes. A total of 436, 70, 551, 993, and 1216 common DEGs between CY6203 and both of its parents were identified at the same stage in panicles and flag leaves. Of the common DEGs, the numbers of upregulated DEGs between CY6203 and CH3203 were all greater than those of upregulated DEGs between CY6203 and C106B in panicles and flag leaves at the booting, flowering, and middle filling stages. Approximately 40.61% of mRNA editing ratios were between 0.4 and 0.6, and 1.68% of mRNA editing events (editing ratio ≥ 0.8) in CY6203 favored one of its parents at three stages or a particular stage, suggesting that the hypothetical heterosis mechanism of CY6203 might involve dominance or epistasis. Also 15,934 DEGs were classified into 19 distinct modules that were classified into three groups by the weighted gene coexpression network analysis. Through transcriptome analysis of panicles and flag leaves in the yield and quality stages, the DEGs in the green-yellow module primarily contributed to the increase in the source of CY6203 due to an in increase in photosynthetic efficiency and nitrogen utilization efficiency, and a small number of DEGs related to the grain number added spikelet number per panicle amplified its sink. The balanced expression of the major high-quality alleles of C106B and CH3203 in CY6203 contributed to the outstanding quality of CY6203. Our transcriptome and genome analyses offer a new data set that may help to elucidate the molecular mechanism governing the yield heterosis and high quality of a hybrid rice variety.

Single-nucleotide polymorphism (SNP) genotyping using cationic conjugated polymers in homogeneous solution

2009 ◽  
Vol 4 (6) ◽  
pp. 984-991 ◽  
Author(s):  
Xinrui Duan ◽  
Wei Yue ◽  
Libing Liu ◽  
Zhengping Li ◽  
Yuliang Li ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Conjugated Polymers ◽  
Homogeneous Solution ◽  
Snp Genotyping ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

scholarly journals Single-Nucleotide-Polymorphism Genotyping ofCoxiella burnetiiduring a Q Fever Outbreak in The Netherlands

2011 ◽  
Vol 77 (6) ◽  
pp. 2051-2057 ◽  
Author(s):  
Cornelis J. J. Huijsmans ◽  
Jeroen J. A. Schellekens ◽  
Peter C. Wever ◽  
Rudolf Toman ◽  
Paul H. M. Savelkoul ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
The Netherlands ◽  
Q Fever ◽  
Snp Genotyping ◽  
Cow Milk ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Animal Reservoir ◽  
Single Nucleotide ◽  
Outbreak Area

ABSTRACTCoxiella burnetiiis the etiological agent of Q fever. Currently, the Netherlands is facing the largest Q fever epidemic ever, with almost 4,000 notified human cases. Although the presence of a hypervirulent strain is hypothesized, epidemiological evidence, such as the animal reservoir(s) and genotype of theC. burnetiistrain(s) involved, is still lacking. We developed a single-nucleotide-polymorphism (SNP) genotyping assay directly applicable to clinical samples. Ten discriminatory SNPs were carefully selected and detected by real-time PCR. SNP genotyping appeared to be highly suitable for discrimination ofC. burnetiistrains and easy to perform with clinical samples. With this new method, we show that the Dutch outbreak is caused by at least 5 differentC. burnetiigenotypes. SNP typing of 14 human samples from the outbreak revealed the presence of 3 dissimilar genotypes. Two genotypes were also present in livestock at 9 farms in the outbreak area. SNP analyses of bulk milk from 5 other farms, commercial cow milk, and cow colostrum revealed 2 additional genotypes that were not detected in humans. SNP genotyping data from clinical samples clearly demonstrate that at least 5 differentC. burnetiigenotypes are involved in the Dutch outbreak.

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