Long PCR amplification of the entire mitochondrial genome from individual helminths for direct sequencing

Abstract

Exploring mitochondrial (mt) genomes has significant implications for various fundamental research areas, including mt biochemistry and physiology, and, importantly, such genomes provide a rich source of markers for population genetics and systematic studies. Although some progress has been made, there is a paucity of information on mt genomes for many metazoan organisms, particularly invertebrates such as parasitic helminths, which relates mainly to the technical limitations associated with sequencing from tiny amounts of material. In this article, we describe a practical long PCR approach for the amplification and subsequent sequencing of the entire mt genome from individual helminths, which overcomes these limitations. The protocol includes the isolation of genomic DNA, long PCR amplification, electrophoresis and sequencing, and takes approximately 1-3 weeks to carry out. The present user-friendly, cost-effective approach has demonstrated utility to the study of a range of parasites, and has the potential to be applied to a wide range of organisms.

The complete mitochondrial genome of Gastrothylax crumenifer (Gastrothylacidae, Trematoda) and comparative analyses with selected trematodes

“…PCR reactions were carried out under the following condition: 94°C for 5 min, then 35 cycles at 94°C/30 s, and annealed at 50°C/30 s, followed by extension at 72°C/1 min per 1 kb, and a final extension of 72°C/7 min. Then, positive PCR amplicons were cloned into pMD19-T vector (TAKARA) for sequencing (Hu et al 2007). …”

Acquisition of Temozolomide Chemoresistance in Gliomas Leads to Remodeling of Mitochondrial Electron Transport Chain

“…Semiquantitative PCR-Isolation and purification of genomic DNA from U251 and UTMZ cells was performed as previously described (25). mtDNA copy number was determined by semiquantitative PCR as previously described (26).…”

The complete mitochondrial genome of Toxascaris leonina: Comparison with other closely related species and phylogenetic implications

“…Each PCR reaction yielded a single band (of the expected sizes at approximately 9 kb and 6 kb respectively) detected in a 0.8% (w/v) agarose gel upon ethidium-bromide staining (not shown). PCR products were sent to Sangon Company (Shanghai, China) for sequencing using a primer walking strategy (Hu et al, 2007;Liu et al, 2013a).…”