Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Summary

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

Abstract

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

Introduction

The mitochondrial genome exists in the form of small (16.5 kb) circular molecules (mtDNA) present in the innermost compartment of the mitochondria named the matrix and encodes 13 subunits of the mitochondrial respiratory chain, as well as the tRNAs and rRNAs necessary for their translation in situ by the mitochondrial ribosome¹. This genome represents approximately 1% of all the proteins necessary for mitochondrial function, the remainder of which are encoded by the nuclear DNA (nDNA). It is commonly assumed that mitochondria are derived from an endosymbiotic fusion event between an alpha-proteobacterial ancestor and an ancestral eukar....

Protocol

Informed consent was provided for the use of the human 3243A>G cybrid cells and the immortalized m.5024C>T MEFs used in this study. Ethical approval was not required in this instance as the patient cells were not collected at the University of Cambridge. The use of human fibroblasts may, however, require ethical approval. It is highly recommended to follow best practices for PCR setup when preparing the sample DNA for pyrosequencing. Frequent amplification using identical primers can lead to amplicon contamination and introduce bias to the subsequent genotyping if strict separation between the pre-PCR and post-PCR areas is not observed. The pipeline presented ....

Results

This section presents an example optimization of a pyrosequencing assay for a human pathogenic mtDNA mutation, as well as sequencing data from the genotyping of heteroplasmic (m.5024C>T) mouse embryonic fibroblasts (MEFs) treated with mitochondrial zinc finger nucleases (mtZFNs). Optimizing the assay for human cells and comparing two different assays demonstrates how to select the most accurate one, whereas genotyping genetically modified MEF cells in the second example serves as an applied example of detecting heter.......

Discussion

A critical aspect for the success of the protocol is avoiding contaminations, particularly when using low amounts of starting material. It is recommended to use a UV hood and filtered pipette tips when preparing the samples wherever possible, as well as to keep the preamplification and post-amplification areas separate. Blank measurements and samples of known heteroplasmy (such as wild-type DNA) should always be included to be used as benchmarks to check for technical or biological bias.

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Materials

Name	Company	Catalog Number	Comments
KOD Hot Start DNA Polymerase	Sigma-Aldrich	71086
PyroMark Assay Design 2.0	QIAGEN
Pyromark Q48 Absorber Strips	QIAGEN	974912
PyroMark Q48 Advanced CpG Reagents (4 x 48)	QIAGEN	974022
Pyromark Q48 Autoprep	QIAGEN	9002470
PyroMark Q48 Cartridge Set	QIAGEN	9024321
Pyromark Q48 Disks	QIAGEN	974901
Pyromark Q48 Magnetic beads	QIAGEN	974203
PyroMark Q48 Software License (1)	QIAGEN	9024325