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

Kähler C, Didlaukat S, Feller AC, Merz H - Diagn Pathol (2007)

Bottom Line: Most PCR protocols use probes to block the wild-type allele during amplification with more or less satisfying result.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-throughput screening because of its robustness.Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pathology, University of Luebeck, Ratzeburger Allee 160, Luebeck, Germany. kaehler@patho.uni-luebeck.de

ABSTRACT

Background: Human mastocytosis is a heterogeneous disorder which is linked to a gain-of-function mutation in the kinase domain of the receptor tyrosine kinase Kit. This D816V mutation leads to constitutive activation and phosphorylation of Kit with proliferative disorders of mast cells in the peripheral blood, skin, and spleen. Most PCR applications used so far are labour-intensive and are not adopted to daily routine in pathological laboratories. The method has to be robust and working on such different materials like archival formalin-fixed, paraffin-embedded tissue (FFPE) and blood samples. Such a method is introduced in this publication.

Methods: 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.

Results: 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.

Conclusion: 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-throughput screening because of its robustness. Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory.

No MeSH data available.


Related in: MedlinePlus

Testing of point mutated primers. 200 ng of genomic HMC1 DNA was used as a PCR template for testing the primer combinations. Primer combination A fits the most.
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Figure 1: Testing of point mutated primers. 200 ng of genomic HMC1 DNA was used as a PCR template for testing the primer combinations. Primer combination A fits the most.

Mentions: In an initial experiment the most suitable primer combination was determined according to Kwok et al. [7] by testing the three combinations on HMC-1 DNA and tonsil DNA as a wild-type control. Primer combination A fulfilled the criteria of specificity in amplifying only the point mutated allele of HMC-1 (see Fig. 1). B and C showed amplification of the WT control so that they are not useful for this assay. The next question which was addressed was the sensitivity of this PCR based assay. For this reason a dilution series with HMC-1 DNA and tonsil DNA had been done whereas the total DNA concentration of every PCR was 200 ng. The minimum concentration of HMC-1 DNA which was still detectable was 10 ng in a total volume of 200 ng of DNA (see Fig. 2). This means a 1:20 dilution. For further testing of primer combination A on patient's material five blood samples and five FFPE samples were used as a template for PCR. The clinical parameters of all these patients clearly indicate mastocytosis and the question was if the Kit point mutation Asp 816 to Val is the reason for this disease pattern. Due to the heterogeneity of patient's samples the DNA in every PCR was variable. At the end the introduced assay with primer combination (A) is able to detect the point mutation in all samples of formalin-fixed, paraffin-embedded tissue (FFPE) and blood (see Fig. 3).


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

Kähler C, Didlaukat S, Feller AC, Merz H - Diagn Pathol (2007)

Testing of point mutated primers. 200 ng of genomic HMC1 DNA was used as a PCR template for testing the primer combinations. Primer combination A fits the most.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211455&req=5

Figure 1: Testing of point mutated primers. 200 ng of genomic HMC1 DNA was used as a PCR template for testing the primer combinations. Primer combination A fits the most.
Mentions: In an initial experiment the most suitable primer combination was determined according to Kwok et al. [7] by testing the three combinations on HMC-1 DNA and tonsil DNA as a wild-type control. Primer combination A fulfilled the criteria of specificity in amplifying only the point mutated allele of HMC-1 (see Fig. 1). B and C showed amplification of the WT control so that they are not useful for this assay. The next question which was addressed was the sensitivity of this PCR based assay. For this reason a dilution series with HMC-1 DNA and tonsil DNA had been done whereas the total DNA concentration of every PCR was 200 ng. The minimum concentration of HMC-1 DNA which was still detectable was 10 ng in a total volume of 200 ng of DNA (see Fig. 2). This means a 1:20 dilution. For further testing of primer combination A on patient's material five blood samples and five FFPE samples were used as a template for PCR. The clinical parameters of all these patients clearly indicate mastocytosis and the question was if the Kit point mutation Asp 816 to Val is the reason for this disease pattern. Due to the heterogeneity of patient's samples the DNA in every PCR was variable. At the end the introduced assay with primer combination (A) is able to detect the point mutation in all samples of formalin-fixed, paraffin-embedded tissue (FFPE) and blood (see Fig. 3).

Bottom Line: Most PCR protocols use probes to block the wild-type allele during amplification with more or less satisfying result.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-throughput screening because of its robustness.Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pathology, University of Luebeck, Ratzeburger Allee 160, Luebeck, Germany. kaehler@patho.uni-luebeck.de

ABSTRACT

Background: Human mastocytosis is a heterogeneous disorder which is linked to a gain-of-function mutation in the kinase domain of the receptor tyrosine kinase Kit. This D816V mutation leads to constitutive activation and phosphorylation of Kit with proliferative disorders of mast cells in the peripheral blood, skin, and spleen. Most PCR applications used so far are labour-intensive and are not adopted to daily routine in pathological laboratories. The method has to be robust and working on such different materials like archival formalin-fixed, paraffin-embedded tissue (FFPE) and blood samples. Such a method is introduced in this publication.

Methods: 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.

Results: 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.

Conclusion: 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-throughput screening because of its robustness. Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory.

No MeSH data available.


Related in: MedlinePlus