Pyrosequencing: Powerful and Quantitative Sequencing Technology

Kreutz, Martin; Hochstein, Norbert; Kaiser, Julia N.; Narz, Frank; Peist, Ralf

doi:10.1002/0471142727.mb0715s104

Cited by 10 publications

(5 citation statements)

References 29 publications

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“…Apyrase is used to degrade any unincorporated dNTP before adding the next one. A more detailed protocol for pyrosequencing can be found in Kreutz et al., 2013 (Kreutz et al., 2013). Pyrosequencing is currently used for targeted studies, but has partly been replaced by newer methods due to its complicated protocol and need for specialized equipment.…”

Section: General Genotyping Methodsmentioning

confidence: 99%

Overview of Genotyping Technologies and Methods

2023

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Genetics is a cornerstone of molecular biology, and there have been significant developments in genotyping technologies during the last decades. Genotyping can be used for a wide range of applications, such as genealogy, assessing risks and causes for common diseases and health conditions, animal and human research, and forensic investigations. So how do you perform a genetic study? This overview covers key concepts in genetics, the development of common genotyping methods, and a comparison of several techniques, including PCR, microarrays, and sequencing. A general process of the steps involved in genotyping, from DNA preparation to quality control, is described with relevant protocols referenced. Different types of DNA variants are illustrated, including mutations, SNP, insertions, deletions, microsatellites, and copy number variations, with examples of their involvement in disease. We discuss the utilities of genotyping, such as medical genetics, genome‐wide association studies (GWAS), and forensic science. We also provide tips for quality control, analysis, and results interpretation to help the reader design and perform a genetic study or scrutinize such studies from the literature. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

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Section: General Genotyping Methodsmentioning

confidence: 99%

Overview of Genotyping Technologies and Methods

2023

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“…The level of DNA methylation was measured using bisulfite pyrosequencing technology which combined sodium bisulfite DNA conversion chemistry (EZ DNA Methylation-Gold™ kit), polymerase chain reaction (PCR) amplification (Zymo Taq™ PCR PreMix; both from Zymo Research Corporation, Irvine, CA, USA) and sequencing by synthesis assay (Pyromark Gold Q24 Reagents; Qiagen, Hilden, Germany) as previously described (22). A fragment containing 7 CpG sites from OPRK1 promoter was used to represent the DNA methylation of OPRK1 promoter.…”

Section: Methodsmentioning

confidence: 99%

Hypermethylation of the κ1 opioid receptor promoter in Chinese heroin and methamphetamine addicts

Liu

et al. 2018

Exp Ther Med

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Heroin and methamphetamine (METH) addiction continues to be a major social, economic and therapeutic problem worldwide. The opioid pathway may mediate the effects of addictive drugs. However, the potential correlation between the κ1 opioid receptor (OPRK1) and drug addiction has not yet been characterized. The aim of the present study was to investigate the potential association between methylation of the OPRK1 promoter and substance abuse. Bisulfite pyrosequencing technology was used to determine the levels of OPRK1 promoter methylation in 60 drug abusers (30 heroin and 30 METH addicts) and 52 controls, observed to exhibit no significant differences in age or gender. The results indicated that levels of OPRK1 promoter methylation were significantly higher in drug addicts when compared with controls (P=2.43×10−4). Significant correlations between OPRK1 promoter methylation and the length and frequency of drug use were also observed in male heroin addicts (length: r=0.661, P=0.007; frequency: r=−0.684, P=0.005). In addition, a luciferase reporter gene assay indicated that the OPRK1 promoter fragment was able to regulate gene expression (fold change between two groups >32.12, P≤0.0001). In conclusion, results of the present study indicate that methylation of the OPRK1 promoter contributes to the pathophysiology of drug addiction.

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“…Either the forward or the reverse primer is modified with a biotin molecule on its 5′ end [20]. The sequencing primer is often designed in a reverse orientation to the biotinylated strand and is complementary to it [21]. Although amplicons can be relatively long (e.g., 400 bp), sequencing efficiency drops after 140 base pairs.…”

Section: Assay Design and Considerations For A Successful Pyrosequenc...mentioning

confidence: 99%

Pyrosequencing: Current forensic methodology and future applications—a review

et al. 2022

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The recent development of small, single-amplicon-based benchtop systems for pyrosequencing has opened up a host of novel procedures for applications in forensic science. Pyrosequencing is a sequencing by synthesis technique, based on chemiluminescent inorganic pyrophosphate detection. This review explains the pyrosequencing workflow and illustrates the step-by-step chemistry, followed by a description of the assay design and factors to keep in mind for an exemplary assay. Existing and potential forensic applications are highlighted using this technology. Current applications include identifying species, identifying bodily fluids, and determining smoking status. We also review progress in potential applications for the future, including research on distinguishing monozygotic twins, detecting alcohol and drug abuse, and other phenotypic characteristics such as diet and body mass index. Overall, the versatility of the pyrosequencing technologies renders it a useful tool in forensic genomics.

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Pyrosequencing: Powerful and Quantitative Sequencing Technology

Cited by 10 publications

References 29 publications

Overview of Genotyping Technologies and Methods

Overview of Genotyping Technologies and Methods

Hypermethylation of the κ1 opioid receptor promoter in Chinese heroin and methamphetamine addicts

Pyrosequencing: Current forensic methodology and future applications—a review

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