Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping

Royo, José Luis; Hidalgo, Manuel; Ruiz, Agustı́n

doi:10.1038/nprot.2007.244

Cited by 104 publications

(90 citation statements)

References 19 publications

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“…Target genomic DNA fragments were amplified by polymerase chain reaction (PCR) using the primers detailed in Supporting information, Table S1; 5 pmol each of the forward and reverse primers, one being biotinylated, were employed to amplify the fragments of interest in a final reaction volume of 12 ll. PCR products were rendered single-stranded according to an established protocol [21]. Three pmol of the sequencing primers provided in Supporting information, Table S1 were used in each case to carry out pyrosequencing of the amplified fragments on the PyroMark Q96 MD apparatus (Qiagen).…”

Section: Pyrosequencingmentioning

confidence: 99%

Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency

Elgizouli

Lowe

Speckmann

et al. 2015

Clinical and Experimental Immunology

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SummaryThe gene PIK3CD codes for the catalytic subunit of phosphoinositide 3-kinase d (PI3Kd), and is expressed solely in leucocytes. Activating mutations of PIK3CD have been described to cause an autosomal dominant immunodeficiency that shares clinical features with common variable immunodeficiency (CVID). We screened a cohort of 669 molecularly undefined primary immunodeficiency patients for five reported mutations (four gain-of-function mutations in PIK3CD and a loss of function mutation in PIK3R1) using pyrosequencing. PIK3CD mutations were identified in three siblings diagnosed with CVID and two sporadic cases with a combined immunodeficiency (CID). The PIK3R1 mutation was not identified in the cohort. Our patients with activated PI3Kd syndrome (APDS) showed a range of clinical and immunological findings, even within a single family, but shared a reduction in naive T cells. PIK3CD gain of function mutations are more likely to occur in patients with defective B and T cell responses and should be screened for in CVID and CID, but are less likely in patients with a pure B cell/hypogammaglobulinaemia phenotype.

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Section: Pyrosequencingmentioning

confidence: 99%

Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency

Elgizouli

Lowe

Speckmann

et al. 2015

Clinical and Experimental Immunology

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“…LINE1%5mC levels were quantified using pyrosequencing (EpigenDx). 27 Methylation levels were examined at four CpG sites in the LINE1 promoter (−492 to -419 bp from ATG). Each 50 μl PCR contained the bisulfite-treated DNA, 10× PCR buffer, 3.0 mM MgCl 2, 200 μM dNTPs, 0.2 µM primers, 1.25 U DNA polymerase (HotStar, Qiagen Inc.).…”

Section: Line1 Methylation Quantificationmentioning

confidence: 99%

“…Each 50 μl PCR contained the bisulfite-treated DNA, 10× PCR buffer, 3.0 mM MgCl 2, 200 μM dNTPs, 0.2 µM primers, 1.25 U DNA polymerase (HotStar, Qiagen Inc.). A biotinylated primer was used to capture one single-stranded DNA template for pyrosequencing 27,28 using the Pyrosequencing PSQ96 HS System (Biotage). One T/C SNP per locus was evaluated using QCpG software (Biotage).…”

Section: Line1 Methylation Quantificationmentioning

confidence: 99%

LINE1methylation levels associated with increased bladder cancer risk in pre-diagnostic blood DNA among US (PLCO) and European (ATBC) cohort study participants

et al. 2013

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“…The analysis was constructed based on the PyroMark Q96 ID user manual and pyrosequencing troubleshooting criteria from nature protocol [12][13][14] .…”

Section: Discussionmentioning

confidence: 99%

Optimization of Pyrosequencing Method to Detect IVS1-NT5 ß-Globin Gene Mutation in ß-Thalassemia

Maskoen¹,

Sofiana²,

Reniarti³

2017

IJPTR

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: β-thalassemia incidence in Indonesia is reaching 2.500 cases per year, so that it is still a threat for Indonesian's society due to the high therapeutic cost. Prenatal diagnosis for detecting β-globin gene mutation is important to be developed in order to decrease β-thalassemia incidence. Pyrosequencing, a new DNA sequencing method, owns the ability to detect DNA mutation quickly for prenatal diagnosis importance. A specific β-globin gene mutation for Indonesian population is needed to make the mutation detection process become more effective, which is the mutation in the intervening sequence 1 nucleotide 5 (IVS1-NT5) region. Hence, the objective of this study is to know the optimal pyrosequencing condition to detect the IVS1-NT5 β-globin gene mutation in β-thalassemia. The sample used was the stored DNA material which the mutation had been detected using polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) for pyrosequencing optimization. There were two steps of optimization, both in the amplification step using polymerase chain reaction (PCR), and pyrosequencing step. The obtained result in amplification step was 55 o C for primer annealing temperature. In pyrosequencing step, the enzyme and substrate storage condition, cartridge's needle performance, and quality of sequencing primer were observed as factors which influence peak formation in the pyrograms, Nevertheless, due to the absence of the proper peaks on the pyrograms of this study, the pyrosequencing method to detect IVS1-NT5 is not yet optimized.

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Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping

Cited by 104 publications

References 19 publications

Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency

Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency

LINE1methylation levels associated with increased bladder cancer risk in pre-diagnostic blood DNA among US (PLCO) and European (ATBC) cohort study participants

Optimization of Pyrosequencing Method to Detect IVS1-NT5 ß-Globin Gene Mutation in ß-Thalassemia

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