scholarly journals Determination of Single-Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing

2000 ◽  
Vol 10 (8) ◽  
pp. 1249-1258 ◽  
Author(s):  
A. Alderborn
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Dna Sequencing ◽  
Real Time ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

scholarly journals Homogeneous Real-Time Detection of Single-Nucleotide Polymorphisms by Strand Displacement Amplification on the BD ProbeTec ET System

2003 ◽  
Vol 49 (10) ◽  
pp. 1599-1607 ◽  
Author(s):  
Sha-Sha Wang ◽  
Keith Thornton ◽  
Andrew M Kuhn ◽  
James G Nadeau ◽  
Tobin J Hellyer
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Dna Sequencing ◽  
Real Time ◽  
Whole Blood ◽  
Nucleotide Polymorphisms ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Single Nucleotide ◽  
Buccal Swabs ◽  
Real Time Detection

Abstract Background: The BD ProbeTec™ ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations. Method: The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human β2-adrenergic receptor (β2AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing. Results: Unprocessed whole blood was successfully genotyped with as little as 0.1–1 μL of sample per reaction. All six β2AR assays were able to accommodate ≥20 μL of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six β2AR assays agreed with DNA sequencing results. Conclusion: SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.

Development of a simple method for the determination of single nucleotide polymorphisms

2010 ◽  
Vol 34 (8) ◽  
pp. S75-S75
Author(s):  
Weifeng Zhu ◽  
Zhuoqi Liu ◽  
Daya Luo ◽  
Xinyao Wu ◽  
Fusheng Wan
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Nucleotide Polymorphisms ◽  
Simple Method ◽  
Single Nucleotide

scholarly journals Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay

2008 ◽  
Vol 54 (2) ◽  
pp. 406-413 ◽  
Author(s):  
Weston C Hymas ◽  
Wade K Aldous ◽  
Edward W Taggart ◽  
Jeffery B Stevenson ◽  
David R Hillyard
Keyword(s):  
Sequence Analysis ◽  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Nucleotide ◽  
The Real ◽  
Nontranslated Region

Abstract Background: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5′ nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. Methods: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay–like plate end detection. Results: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. Conclusions: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.

Loss of heterozygosity testing using real-time PCR analysis of single nucleotide polymorphisms

2005 ◽  
Vol 132 (3) ◽  
pp. 200-204 ◽  
Author(s):  
Tamara Čačev ◽  
Mladen Jokić ◽  
Radan Spaventi ◽  
Krešimir Pavelić ◽  
Sanja Kapitanović
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Loss Of Heterozygosity ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms

2004 ◽  
Vol 18 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Kenneth Petersen ◽  
Ulla Vogel ◽  
Eszter Rockenbauer ◽  
Kirsten Vang Nielsen ◽  
Steen Kølvraa ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Real Time ◽  
Real Time Pcr ◽  
Molecular Beacons ◽  
Nucleotide Polymorphisms ◽  
Allelic Discrimination ◽  
Single Nucleotide

scholarly journals Analysis of the genetic diversity of Phalaenopsis orchids with single nucleotide polymorphisms and snap markers derived from the Pto gene

2021 ◽  
Vol 53 (4) ◽  
pp. 620-631
Keyword(s):  
Genetic Diversity ◽  
Single Nucleotide Polymorphisms ◽  
Genomic Sequence ◽  
Nucleotide Polymorphisms ◽  
Multiple Sequence ◽  
Functional Link ◽  
Single Nucleotide ◽  
Functional Changes ◽  
Interspecies Variability

The Pto gene is a plant gene that has been reported to be involved in resistance to bacterial pathogens. A partial genomic sequence corresponding to Pto (~449 bp) was isolated from 16 species and four hybrids of Phalaenopsis during 2017 at the Department of Agronomy and Horticulture, IPB University, Bogor, Indonesia. Multiple sequence analysis was performed to find putative single nucleotide polymorphisms (SNPs) and design the corresponding single nucleotide-amplified polymorphism (SNAP) markers, which were in turn used to estimate the genetic diversity of 25 Phalaenopsis species. In total, 20 SNPs, of which 14 were nonsynonymous, were identified from the partial Pto sequences. Eighteen SNAP primers were then developed based on these 14 nonsynonymous and four synonymous SNPs. Validation results showed that 15 SNAP primers showed a polymorphism information content exceeding 0.3, suggesting the existence of more than two alleles for this locus. Upon their use, the SNAP markers described 86% of all interspecies variability. The Pto 52, Pto 349, Pto 229, and Pto 380 SNAP markers were very informative in the determination of genetic diversity. Notably, the existence of these nonsynonymous SNPs implied the possibility of functional changes within the amino acid sequence of the putative PTO protein. Thus, the resulting differences in the activity of the PTO protein may be used to breed tolerance to pathogen infection. Further work may be required to establish a functional link between tolerance to pathogens and the presence of Pto-SNAP markers in Phalaenopsis properly.

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