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Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
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Analysis of Mutational Hotspots in Routinely Processed Bone Marrow Trephines by Pyrosequencing®
Stephan Bartels1, Ulrich Lehmann
1Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|June 25, 2015
Summary
Pyrosequencing enables sensitive detection of single-nucleotide variations in fragmented DNA from bone marrow trephines. This method is cost-effective for analyzing formalin-fixed, paraffin-embedded samples, aiding in diagnosing conditions like myeloproliferative neoplasms.
Area of Science:
- Molecular Pathology
- Genetics
- Biochemistry
Background:
- Formalin-fixed, paraffin-embedded (FFPE) bone marrow trephines preserve morphology but yield fragmented DNA, challenging sequencing applications.
- Fragmented DNA limits amplification product size, posing a significant hurdle for comprehensive genetic analysis in pathology.
- Accurate genetic profiling is crucial for diagnosing and understanding diseases involving bone marrow.
Purpose of the Study:
- To outline a protocol for detecting single-nucleotide variations (SNVs) in FFPE bone marrow trephines using Pyrosequencing.
- To provide guidelines for designing Pyrosequencing assays and interpreting Pyrograms for FFPE samples.
- To demonstrate the utility of Pyrosequencing for SNV detection in myeloproliferative neoplasms (MPNs).
Main Methods:
- DNA isolation from FFPE bone marrow trephines.
- Pyrosequencing assay design and optimization for fragmented DNA.
- Quantitative analysis of SNVs using Pyrosequencing and Pyrogram evaluation.
Main Results:
- Pyrosequencing provides a sensitive and quantitative method for SNV detection in FFPE bone marrow DNA.
- The 96-well-plate format offers a cost-effective, medium-throughput solution for genetic analysis.
- Successful application demonstrated in the context of myeloproliferative neoplasms.
Conclusions:
- Pyrosequencing is a robust technique for SNV detection in challenging FFPE bone marrow samples.
- This method facilitates genetic profiling crucial for diagnosing hematological malignancies.
- The outlined protocol and guidelines support the broader application of Pyrosequencing in diagnostic pathology.
