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(New page: [http://www.nature.com/nature/journal/v464/n7288/pdf/nature08802.pdf Hereoplastic mitochondrial DNA mutations in normal and tumor cells] by Yiping He ''et al.'' ==Background== ===Mitochon...)
 
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===Capture-Based mtDNA Enrichment Strategy===
===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.
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.


==Results==
==Results==

Latest revision as of 05:06, 10 April 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.

Results

First the researchers sequenced the mitochondrial DNA from colon mucousae by methods listed above. To control for polymerase errors, they made and sequenced a DNA library of genomic colon mucousal DNA and found that no base was mutated more than 0.82% of the time so they assumed that any mutation that occurred 1.6% of the time in their mtDNA sequencing was an actual mutations, and not an artifact.

In their screen they identified 28 homoplasmic alleles and 8 heteroplasmic alleles in normal human colonic mucousa. After sequencing nine additionals samples it was determined that, on average, a sample contained all 28 homoplasmic variants and four heteroplasmic varients.

They then sampled ten other tissues from a second patient including brain, heart, skeletal muscle, and lung.

They came up with three possible reasons for these vartions:

  1. These varients represent mtDNA inherited from the father.
  2. These variations are inherited from the mother with bottlenecks during development causing tissue specific variations
  3. These variations represent new mutations that occur during embryotic development.

In order to test these theories, researchers analyzed lymphocte mtDNA from two families. The found that of heteroplasmic variants found in the fathers and not in the mothers, none were found in children; this result confirms that mitochondrial DNA is strictly maternally inherited. When analyzing the maternal data for the first family, they found that two heteroplasmic variants found in the mother were found in the children at the same allelic frequency confirming that heteroplasmic variants are maternally inherited. In the second family, the three heteroplasmic varients found in the mother were not found in the children. Researchers concluded that this is likely not due to a developmental bottleneck, but rather that these variants are tissue specific to somatic cells and thus would not be passed to her offspring.

Significance

The research presented provides a new insight into mitochondrial DNA. Results suggest that heteroplasmic variants can be used as biomarkers to not only diagnose cancer but to track its progress through pre- and post-surgery analysis of blood.

The results also give critical information to forensic scientists who often use mtDNA for suspect identification and/or elimination. This study suggests that samples collected from suspects must be from the same tissue type in order to have accurate results because if variation found from tissue to tissue within on individual.

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)