Dataset Information

Role of excess inorganic pyrophosphate in primer-extension genotyping assays.

ABSTRACT: We have developed and genotyped >15,000 SNP assays by using a primer extension genotyping assay with fluorescence polarization (FP) detection. Although the 80% success rate of this assay is similar to those of other SNP genotyping assays, we wanted to determine the reasons for the failures and find ways to improve the assay. We observed that the failed assays fell into three general patterns: PCR failure, excess of heterozygous genotypes, and loss of FP signal for one of the dye labels. After analyzing several hundred failed assays, we concluded that 5% of the assays had PCR failure and had to be redesigned. We also discovered that the other two categories of failures were due to misincorporation of one of the dye-terminators during the primer extension reaction as a result of primer shortening with a reverse reaction involving inorganic pyrophosphate, and to the quenching of R110-terminator after its incorporation onto the SNP primer. The relatively slow incorporation of R110 acycloterminators by AcycloPol compounds the problem with the R110 label. In this report, we describe the source of the problems and simple ways to correct these problems by adding pyrophosphatase, using quenching as part of the analysis, and replacing R110 by Texas red as one of the dye labels. With this new protocol, we have achieved approximately 95% success rate in assay development without the need for optimization.

SUBMITTER: Xiao M

PROVIDER: S-EPMC515321 | biostudies-literature | 2004 Sep

REPOSITORIES: biostudies-literature

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Role of excess inorganic pyrophosphate in primer-extension genotyping assays.

Xiao Ming M Phong Angie A Lum Kristen L KL Greene Richard A RA Buzby Philip R PR Kwok Pui-Yan PY

Genome research 20040812 9

We have developed and genotyped >15,000 SNP assays by using a primer extension genotyping assay with fluorescence polarization (FP) detection. Although the 80% success rate of this assay is similar to those of other SNP genotyping assays, we wanted to determine the reasons for the failures and find ways to improve the assay. We observed that the failed assays fell into three general patterns: PCR failure, excess of heterozygous genotypes, and loss of FP signal for one of the dye labels. After an ...[more]

PMID: 15310656

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Project description:Excess PPi triggers developmental defects in a cell-autonomous manner. The level of inorganic pyrophosphate (PPi) must be tightly regulated in all kingdoms for the proper execution of cellular functions. In plants, the vacuolar proton pyrophosphatase (H+-PPase) has a pivotal role in PPi homeostasis. We previously demonstrated that the excess cytosolic PPi in the H+-PPase loss-of-function fugu5 mutant inhibits gluconeogenesis from seed storage lipids, arrests cell division in cotyledonary palisade tissue, and triggers a compensated cell enlargement (CCE). Moreover, PPi alters pavement cell (PC) shape, stomatal patterning, and functioning, supporting specific yet broad inhibitory effects of PPi on leaf morphogenesis. Whereas these developmental defects were totally rescued by the expression of the yeast soluble pyrophosphatase IPP1, sucrose supply alone canceled CCE in the palisade tissue but not the epidermal developmental defects. Hence, we postulated that the latter are likely triggered by excess PPi rather than a sucrose deficit. To formally test this hypothesis, we adopted a spatiotemporal approach by constructing and analyzing fugu5-1 PDF1 pro ::IPP1, fugu5-1 CLV1 pro ::IPP1, and fugu5-1 ICL pro ::IPP1, whereby PPi was removed specifically from the epidermis, palisade tissue cells, or during the 4 days following seed imbibition, respectively. It is important to note that whereas PC defects in fugu5-1 PDF1 pro ::IPP1 were completely recovered, those in fugu5-1 CLV1 pro ::IPP1 were not. In addition, phenotypic analyses of fugu5-1 ICL pro ::IPP1 lines demonstrated that the immediate removal of PPi after seed imbibition markedly improved overall plant growth, abolished CCE, but only partially restored the epidermal developmental defects. Next, the impact of spatial and temporal removal of PPi was investigated by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF MS). Our analysis revealed that the metabolic profiles are differentially affected among all the above transgenic lines, and consistent with an axial role of central metabolism of gluconeogenesis in CCE. Taken together, this study provides a conceptual framework to unveil metabolic fluctuations within leaf tissues with high spatio-temporal resolution. Finally, our findings suggest that excess PPi exerts its inhibitory effect in planta in the early stages of seedling establishment in a tissue- and cell-autonomous manner.

Dataset Information

Role of excess inorganic pyrophosphate in primer-extension genotyping assays.

Publications

Role of excess inorganic pyrophosphate in primer-extension genotyping assays.

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