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Old 12-12-2012, 11:11 AM   #1
lethalfang
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Default A few basic questions about mitochondrial DNA resequencing

Hi guys,

I've a few questions about looking for mutations in human mitochondrial DNA.

1) Since there are only 17 kbp of DNA in the mitochondria, if I can purify mitochondrial DNA, get rid of the nuclear DNA, I don't need any capture kits to sequence mitochondrial DNA. Is that correct?

2) Are there any companies that make primers/oligos that target mitochondrial genes? For instance, Agilent HaloPlex does not.

3) Is it possible to capture some genes using the typical "Oncogene Panel" or a customized panel, and then combine with the whole mitochondrial DNA as in "Step 1," and sequence the combined customized nuclear gene and mitochondrial DNA?

The answers should be the same for both MiSeq and Ion Torrent, correct?

Thanks in advance.
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Old 12-17-2012, 07:00 AM   #2
ngseq
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1. In theory, you can. But it may be a problem to isolate mtDNA with sufficient amount or purity for library prep in practice. It seems that most labs use long range PCR.

2. I don't know the answer. But I guess you can certainly design your own primers using primer3. Just be aware of nuclear mitochondrial pseudogenes when doing so.

3. I think you can combine the two as long as you can figure out the correct ratio as this is going to determine the relative coverage between them.
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Old 01-03-2013, 11:23 AM   #3
csquared
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The PCR primers used by Affymetrix to amplify the mtDNA in two overlapping fragments for use on their sequencing array work great for amplification prior to library prep and sequencing. The fragments are a bit over 8Kb so you still need a traditional library prep but after pooling the fragments in molar equivalents, treat the samples like standard genomic DNA and everything works great.

There are a few different ways to purify mtDNA away from nuclear, but all are pretty labor intensive compared to PCR. It is possible through if you really want to use an non-amplified approach.

mtDNA sequences are pretty abundant and it is not unusual to see them carry through on capture experiments like exome sequencing. You could certainly intentionally capture the mtDNA with some optimization.
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