CH391L/S2013 Taylor Pursell April 3 2013

Hereoplastic mitochondrial DNA mutations in normal and tumor cells by Yiping He et al.

Background

Mitochondria

Mitochondria is a membrane enclose organelle found in most eukaryotic cells. They were first visualized in the 1950s visualized in chick cells with electron microscopy [1]. Mitochondria generate most of the cell’s ATP; they are also involed in signaling, differentiation, cell death, control of cell cycle and cell growth [2]. All mitochondria share a few characteristics included double lipid membrane and inner membrane folds called cristae. Although the mitochondria first visualized were bean shaped, further research has revealed that mitochondrial morphology varies greatly between cell types and even within the same cell [1].

The number of mitochondria contained in a ranges from 50 to 10,000 depending on the tissue's need for energy. For example, cells which make up the heart, skeletal muscle, brain, and eye have the highest number of mitochondria in the human body containing up to 10,000 mitochondria per cell [2].

Mitochondrial Genome

Most mitochondrial DNA, including the human mtDNA, is double stranded and circular. In unicellular and more rarely in multicellular organism, it is linear [4]. The human mitochondrial genome is roughly 16 kilobases in length and codes for 37 genes including cytochrome b, 12s rRNA, 16s rRNA, and NADH dehydrogenases among others. Each mitochondrion contains five to ten copies of the its genomes [5].

Introduction

Although in the past mtDNA has been considered to be homogeneous within an organism, but this study reveals widespread variation in the mitochondrial genome in normal cells throughout the human body. The amount of variation, or heteroplasmy, varies not only from one tissue to another but also within one cell type. They conclude that humans are a related mixture of mitochondrial genotypes. In this study, they explore this heteroplasmy in wildtype and cancer cells [3].

Methods

The most important method in this paper is the sequencing of mtDNA collected from different tissues and different individuals accurately enough to compare point mutations which make up the heteroplasmy.

PCR-Based mtDNA Enrichment Strategy

Three different sets of PCR primer sets were designed to amplify the whole mitochondrial genome. Two set have small amplicons while the third has larger amplicons. These PCR products are purified, then blunted before being ligated back together to recreated the mtDNA. This ligated product is them fragmented again using sonication. Following the Illumina protocol for library preparation, they adenylated and added adaptors to the ends of the fragments. These fragments could then be sequenced-by-synthesis.

Capture-Based mtDNA Enrichment Strategy

The other strategy uses PCR amplification to selectively amplify mtDNA from colon mucousal DNA. They then used these products as the templates to amplify with biotinylated primers to create biotinylated PCR products which they denatured to make 50 biotinylated ssDNA probes for the mitochondial genome. They then incubated these probes with a ssDNA library made from the total colon mucousal DNA allowing for hybridization. Purification was done using strepavidin-coated beads, allowing them to isolate only mtDNA. They did a second hybridization and pruification using the fragments isolated from the first round in order to give them a very pure sample. Finally they used PCR to amplify the fragments isolated, attached adaptors (see above), and sequences them by sequencing-by-synthesis.

References

1. Detmer, S. A. and Chan, D. C. Functions and dysfunctions of mitochondrial dynamics. Nature 8: 870-879 (2007)
2. McBride, H. M., Neuspiel, M., Wasiak, S. Mitochondria: more than just a powerhouse. Current Biology 16: R551-R560 (2006)
3. He, Y., et al. Heteroplasmic mitochondrial DNA mutations in normal and tumour cells. Nature 464: 610-614 (2010)
4. Nosek J, Tomáska L, Fukuhara H, Suyama Y, Kovác L (May 1998). "Linear mitochondrial genomes: 30 years down the line". Trends Genet. 14 (5): 184–8.
5. Maechler, R., Wollheim, C. B. Mitochondrial function in normal and diabetic β-cells. Nature 414: 807-812 (2001)