Pyrosequencing: History, biochemistry and future

Ahmadian, Afshin; Ehn, M.; Hober, Sophia

doi:10.1016/j.cccn.2005.04.038

Cited by 294 publications

(184 citation statements)

References 109 publications

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“…We chose to use the method of pyrosequencing because it allows high-throughput scoring of SNPs and the technology is affordable for an individual laboratory (reviewed by Ronaghi 2001;Fakhrai-Rad et al 2002;Langaee and Ronaghi 2005;Ahmadian et al 2006). Briefly, the pyrosequencing method involves three steps.…”

Section: Resultsmentioning

confidence: 99%

Identification of Mutations in Caenorhabditis elegans That Cause Resistance to High Levels of Dietary Zinc and Analysis Using a Genomewide Map of Single Nucleotide Polymorphisms Scored by Pyrosequencing

Bruinsma¹,

Schneider

Davis

et al. 2008

Genetics

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Zinc plays many critical roles in biological systems: zinc bound to proteins has structural and catalytic functions, and zinc is proposed to act as a signaling molecule. Because zinc deficiency and excess result in toxicity, animals have evolved sophisticated mechanisms for zinc metabolism and homeostasis. However, these mechanisms remain poorly defined. To identify genes involved in zinc metabolism, we conducted a forward genetic screen for chemically induced mutations that cause Caenorhabditis elegans to be resistant to high levels of dietary zinc. Nineteen mutations that confer significant resistance to supplemental dietary zinc were identified. To determine the map positions of these mutations, we developed a genomewide map of single nucleotide polymorphisms (SNPs) that can be scored by the high-throughput method of DNA pyrosequencing. This map was used to determine the approximate chromosomal position of each mutation, and the accuracy of this approach was verified by conducting three-factor mapping experiments with mutations that cause visible phenotypes. This is a generally applicable mapping approach that can be used to position a wide variety of C. elegans mutations. The mapping experiments demonstrate that the 19 mutations identify at least three genes that, when mutated, confer resistance to toxicity caused by supplemental dietary zinc. These genes are likely to be involved in zinc metabolism, and the analysis of these genes will provide insights into mechanisms of excess zinc toxicity.

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Section: Resultsmentioning

confidence: 99%

Identification of Mutations in Caenorhabditis elegans That Cause Resistance to High Levels of Dietary Zinc and Analysis Using a Genomewide Map of Single Nucleotide Polymorphisms Scored by Pyrosequencing

Bruinsma¹,

Schneider

Davis

et al. 2008

Genetics

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“…There are a number of techniques to study the epigenetic DNA methylation pattern, such as BSP [16], pyrosequencing [4], Combined Bisulfite Analysis (COBRA) [41], methylation microarrays [7], MSP [19] or Maldi-TOF mass spectrometry [25], and they have differences in their specificity, sensibility, accuracy and cost. We have recently published two reports identifying by BSP different diet-related epigenetic marks in leptin [29] and TNF-alpha [11] that could be used as biomarkers of weight loss.…”

Section: Discussionmentioning

confidence: 99%

Leptin and TNF-alpha promoter methylation levels measured by MSP could predict the response to a low-calorie diet

et al. 2011

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Obesity-associated adipose tissue enlargement is characterized by an enhanced proinflammatory status and an elevated secretion of adipokines such as leptin and cytokines such as TNF-alpha. Among the different mechanisms that could underlie the interindividual differences in obesity, epigenetic regulation of gene expression has emerged as a potentially important determinant. Therefore, twenty-seven obese women (age: 32-50 years; baseline Body Mass Index, BMI: 34.4±4.2 Kg/m 2 ) were prescribed an eightweek Low-Calorie-Diet and epigenetic marks were assessed. Baseline and endpoint anthropometric parameters were measured and blood samples were drawn. Genomic DNA and RNA from adipose tissue biopsies were isolated before and after the dietary intervention. Leptin and TNF-alpha promoter methylation were measured by MSP after bisulfite treatment and gene expression was also analyzed.Obese women with a successful weight loss (≥5% of initial body weight, n=21) improved the lipid profile and fat mass percentage (-12%, p<0.05). Both systolic (-5%, p<0.05) and diastolic (-8%, p<0.01) blood pressures significantly decreased. At baseline women with better response to the dietary intervention showed lower promoter methylation levels of leptin (-47%, p<0.05) and TNF-alpha (-39%, p=0.071) than the non-responder group (n=6), while no differences were found between responder and non-responder group in leptin and TNF-alpha gene expression analysis. These data suggest that leptin and TNF-alpha methylation levels could be used as epigenetic biomarkers concerning the response to a Low-CalorieDiet. Indeed, methylation profile could help to predict the susceptibility to weight loss as well as some comorbidities such as hypertension or type 2 diabetes.

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“…Many high-throughput SNP genotyping methods have been developed including SNP microarrays [1], MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time of Flight) [2], TaqMan probes [3], PCR resequencing [4], and others [5-7]; however, most of them come at a price. This is that they require expensive machinery and are unsuitable for the kind of small-scale genotyping that is routinely undertaken in most regular laboratories.…”

Section: Introductionmentioning

confidence: 99%

SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping

et al. 2010

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BackgroundPCR-restriction fragment length polymorphism (RFLP) assay is a cost-effective method for SNP genotyping and mutation detection, but the manual mining for restriction enzyme sites is challenging and cumbersome. Three years after we constructed SNP-RFLPing, a freely accessible database and analysis tool for restriction enzyme mining of SNPs, significant improvements over the 2006 version have been made and incorporated into the latest version, SNP-RFLPing 2.ResultsThe primary aim of SNP-RFLPing 2 is to provide comprehensive PCR-RFLP information with multiple functionality about SNPs, such as SNP retrieval to multiple species, different polymorphism types (bi-allelic, tri-allelic, tetra-allelic or indels), gene-centric searching, HapMap tagSNPs, gene ontology-based searching, miRNAs, and SNP500Cancer. The RFLP restriction enzymes and the corresponding PCR primers for the natural and mutagenic types of each SNP are simultaneously analyzed. All the RFLP restriction enzyme prices are also provided to aid selection. Furthermore, the previously encountered updating problems for most SNP related databases are resolved by an on-line retrieval system.ConclusionsThe user interfaces for functional SNP analyses have been substantially improved and integrated. SNP-RFLPing 2 offers a new and user-friendly interface for RFLP genotyping that can be used in association studies and is freely available at http://bio.kuas.edu.tw/snp-rflping2.

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Pyrosequencing: History, biochemistry and future

Cited by 294 publications

References 109 publications

Identification of Mutations in Caenorhabditis elegans That Cause Resistance to High Levels of Dietary Zinc and Analysis Using a Genomewide Map of Single Nucleotide Polymorphisms Scored by Pyrosequencing

Identification of Mutations in Caenorhabditis elegans That Cause Resistance to High Levels of Dietary Zinc and Analysis Using a Genomewide Map of Single Nucleotide Polymorphisms Scored by Pyrosequencing

Leptin and TNF-alpha promoter methylation levels measured by MSP could predict the response to a low-calorie diet

SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping

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