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Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity.

ABSTRACT: Chromatin can be analysed by assaying its sensitivity to DNase I or other nucleases in purified nuclei. Usually, this is performed by Southern analysis of genomic DNA extracted from nuclease-treated nuclei, a methodology that requires many cells. Applying restriction fragment length polymorphisms (RFLPs), this methodology has been used for parental allele-specific chromatin studies on imprinted mammalian genes. However, such allelic studies are limited by the availability of suitable RFLPs. We therefore developed an alternative, PCR and single strand conformation polymorphism (SSCP)-based assay with which allelic sensitivity to nucleases can be determined in virtually all localised regions that have nucleotide polymorphisms. We also demonstrate that analysis of DNase I sensitivity can be performed on permeabilised cells. Combining the two approaches, in the imprinted mouse U2af1-rs1 gene we analysed parental allele-specific chromatin conformation in limited numbers of cultured cells. We also applied the PCR-SSCP approach to assay allelic DNA methylation at specific restriction enzyme sites. In summary, we developed an allele-specific assay that should be useful for biochemical and developmental investigation of chromatin, in particular for studies on genomic imprinting and X-chromosome inactivation.

SUBMITTER: Gregory RI

PROVIDER: S-EPMC148708 | biostudies-other | 1999 Nov

REPOSITORIES: biostudies-other

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Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity.

Gregory R I RI Feil R R

Nucleic acids research 19991101 22

Chromatin can be analysed by assaying its sensitivity to DNase I or other nucleases in purified nuclei. Usually, this is performed by Southern analysis of genomic DNA extracted from nuclease-treated nuclei, a methodology that requires many cells. Applying restriction fragment length polymorphisms (RFLPs), this methodology has been used for parental allele-specific chromatin studies on imprinted mammalian genes. However, such allelic studies are limited by the availability of suitable RFLPs. We t ...[more]

PMID: 10536165

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Project description:BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.

Dataset Information

Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity.

Publications

Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity.

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