High‐resolution melt analysis of the minisatellite D1S80: A potential forensic screening tool

Pomeroy, Robert S.; Balamurugan, Kuppareddi; Wong, Helena; Duncan, George

doi:10.1002/elps.201400143

Cited by 4 publications

(4 citation statements)

References 35 publications

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“…Like GC‐MS methods, DNA quantitation methods using PCR are widely used with high confidence. Although not widely adopted by crime labs as GC‐MS, several studies have reported new PCR HRM assays for forensic use for human STR and mitochondrial DNA typing, sex determination, DNA quantitation, body fluid analysis, blowfly species identification, and plant drug identification . In summary, this real‐time PCR HRM assay could be used to rapidly detect and identify Psilocybe species DNA even if it is comingled with human and/or marijuana DNA in a trace or degraded sample such as saliva or material recovered from drug paraphernalia.…”

Section: Discussionmentioning

confidence: 99%

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Cowan

Elkins

2017

Journal of Forensic Sciences

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Psilocybe cubensis, or "magic mushroom," is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web-based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR Green I, Radiant™ Green, or LCGreen Plus intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant™ Green dye.

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Section: Discussionmentioning

confidence: 99%

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Cowan

Elkins

2017

Journal of Forensic Sciences

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“…By selecting specific genes for amplification, a targeted HRM-based methodology reduces the sample preparation requirements such that they are similar to those of a multiplexed qPCR. Furthermore, with the development of selective dyes, such as LCGreen, SYTO-9, and EvaGreen, HRM analysis can discriminate between the larger amplicons required for MLST, ranging from ∼600 to 900 bp (16–20). The purpose of this study was to develop and validate the single-nucleotide polymorphism (SNP)-level discriminatory power of HRM to enable rapid and cost-effective differentiation of ST131 E.…”

Section: Introductionmentioning

confidence: 99%

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli

Harrison

Hanson

2017

Antimicrob Agents Chemother

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Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification.

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“…Single Nucleotide Polymorphism SNPs: "Genetic variations for example single nucleotide SNPs (SNPs) are common polymorphism type contained by a population (14).SNPs can be presented in different ways, and since there is currently no consensus, this can lead to confusion.Knowing the Genetic makeup and variations in the genome of an individual or population can help the physicians to understand the influencing factors that an individual has towards certain diseases and may help in directing the medicines." SNP Detection: High-Resolution Melt (HRM): High-Resolution Melt (HRM) analysis is an effective technique by which single nucleotide modifications can be identified in strands of DNA by exploring the level of DNA separation through the temperature at which DNA become denatured (15).…”

Section: Methodsmentioning

confidence: 99%

Association of Genetic Variants rs1888747 and rs10868025 of FRMD3 Gene with Diabetic Nephropathy in Pakistani Patients

Sharif¹,

Hassan²,

Rehman³

et al. 2022

PJMHS

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Background: Diabetic Nephropathy is also known as diabetic kidney disease. The major cause of diabetic nephropathy is diabetes Many candidate genes and genome-wide association studies have proved that gene FRMD3 is highly associated with diabetic nephropathy. Aim of the study: This study aimed to investigate the association of rs1888746 and rs1888747 with diabetic nephropathy in our local population of Pakistan. Research Design: For this study, we enrolled 50 physician-diagnosed patients and 25 healthy control from the young population. Genomic DNA was extracted by organic method and visualized on Agarose gel. Methodology: Monoplex PCR, real-time PCR, genotyping via high-resolution melting curve analysis,and SPSS software were used to accomplish the research work. Results: Our study found that variant rs1888746 shows an insignificant association with (p<0.0982) and rs188747 shows a significant association with (p<0.0498), but results need to be confirmed on a larger scale. Conclusion: The results concluded that our selected rs1888747 on FRMD3 gene shows the allelic association with diabetic nephropathy in Pakistani local population whereas r1888746 did not. Keywords: Genome Wide Association Studies, Diabetic Nephropathy, Genomic DNA, FRMD3 gene

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High‐resolution melt analysis of the minisatellite D1S80: A potential forensic screening tool

Cited by 4 publications

References 35 publications

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli

Association of Genetic Variants rs1888747 and rs10868025 of FRMD3 Gene with Diabetic Nephropathy in Pakistani Patients

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