Methodology article

Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences

Liane Fendt¹ , Bettina Zimmermann¹ , Martin Daniaux² and Walther Parson¹

¹  Institute of Legal Medicine, Innsbruck Medical University, Müllerstrasse 44, Austria

²  Clinical Department of Radiology, Innsbruck Medical University, Austria

author email corresponding author email

BMC Genomics 2009, 10:139doi:10.1186/1471-2164-10-139

Published:	30 March 2009

Abstract

Background

It has been demonstrated that a reliable and fail-safe sequencing strategy is mandatory for high-quality analysis of mitochondrial (mt) DNA, as the sequencing and base-calling process is prone to error. Here, we present a high quality, reliable and easy handling manual procedure for the sequencing of full mt genomes that is also appropriate for laboratories where fully automated processes are not available.

Results

We amplified whole mitochondrial genomes as two overlapping PCR-fragments comprising each about 8500 bases in length. We developed a set of 96 primers that can be applied to a (manual) 96 well-based technology, which resulted in at least double strand sequence coverage of the entire coding region (codR).

Conclusion

This elaborated sequencing strategy is straightforward and allows for an unambiguous sequence analysis and interpretation including sometimes challenging phenomena such as point and length heteroplasmy that are relevant for the investigation of forensic and clinical samples.