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Updated: Apr 8, 2026
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
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.
Abstract:
Formalin-fixed, paraffin-embedded (FFPE) bone marrow trephines are widely used in pathology, because they best preserve the morphological details of the bone marrow. However, DNA isolated from FFPE material is fragmented, limiting the size of amplification products, which is a challenge for all sequencing applications.Pyrosequencing(®) is a quantitative and sensitive method for the detection of single-nucleotide variations (SNVs) in DNA samples. Pyrosequencing can easily be performed in a 96-well-plate format with a cost-effective medium-sized throughput.This chapter provides a general outline of SNV detection in FFPE bone marrow trephines, including a detailed protocol of the Pyrosequencing procedure and guidelines for the design of new assays and evaluation of Pyrograms. The strengths of this approach are discussed using myeloproliferative neoplasms as an example.
Insights
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.
