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Sensitive and reliable detection of Kit point mutation Asp 816 to Val in pathological material

DOI: 10.1186/1746-1596-2-37

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The Kit point mutation Asp 816 to Val is heterozygous which means a problem in detection by PCR because the wild-type allele is also amplified and the number of cells which bear the point mutation is in most of the cases low. Most PCR protocols use probes to block the wild-type allele during amplification with more or less satisfying result. This is why point-mutated forward primers were designed and tested for efficiency in amplification of the mutated allele.One primer combination (A) fits the most for the introduced PCR assay. It was able just to amplify the mutated allele with high specificity from different patient's materials (FFPE or blood) of varying quality and quantity. Moreover, the sensitivity for this assay was convincing because 10 ng of DNA which bears the point mutation could be detected in a total volume of 200 ng of DNA.The PCR assay is able to deal with different materials (blood and FFPE) this means quality and quantity of DNA and can be used for high-througput screening because of its robustness. Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory.1,000 to 8,000 incidences of human mastocytosis are reported every year in the US [1]. Human mastocytosis is characterised by accumulation of mast cells in different organs. It is a heterogenous group of disorders which can be divided into the categories cutaneous mastocytosis (CM) and systemic mastocytosis (SM) which is commonly seen in adults by histological lesions in the bone marrow and other non-cutaneous organs [2,3]. SM can be further divided into the categories indolent systemic mastocytosis (ISM), SM with an associated clonal hematologic non-mast cell lineage disease (AHNMD), aggressive sytemic mastocytosis (ASM), and mast cell leukemia (MCL). ISM is the most common form which involves skin, bone marrow, and GI tract with good prognosis for the patient. First relations between mastocytosis and activating mutations in the receptor tyrosine kin


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