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Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment.

McKernan KJ, Spangler J, Zhang L, Tadigotla V, McLaughlin S, Warner J, Zare A, Boles RG - PLoS ONE (2014)

Bottom Line: We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides.Use of this enzyme-and-nucleotide combination enables what we term a "DNA diode", where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays.Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment.

View Article: PubMed Central - PubMed

Affiliation: Courtagen Life Sciences, Woburn, Massachusetts, United States of America.

ABSTRACT
We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides. Use of this enzyme-and-nucleotide combination enables what we term a "DNA diode", where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays. Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment. These additional nucleotides enable a novel DNA decontamination technique that generates ephemeral and easy to decontaminate DNA.

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Decontamination effectiveness.To measure decontamination potential we mixed equimolar 5me-dCTP amplified mtDNA into non-methylated Target mtDNA. Methylated and non-methylated DNA were from mtDNA haplogroups differing in 8 loci. Each haplogroup mtDNA sample was barcoded with unique DNA barcodes prior to pooling, decontamination and amplification. Complete decontamination was measured via sequencing the mixed libraries to 10,000× coverage and measuring heteroplasmy levels with and without MspJI decontamination. MspJI digestion removed 100% of expected heteroplasmy contaminants(red) suggesting it can decontaminate up to equimolar contamination events. Undigested pooled libraries were sequenced as a control (blue) and exhibited 35–65% heteroplasmy levels. These artificial heteroplasmies were produced by pooling a methylated mitochondrial Long Range PCR product from a different haplogroup into a non methylated product. This haplogroup is completely removed by the decontamination methods described.
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pone-0096492-g005: Decontamination effectiveness.To measure decontamination potential we mixed equimolar 5me-dCTP amplified mtDNA into non-methylated Target mtDNA. Methylated and non-methylated DNA were from mtDNA haplogroups differing in 8 loci. Each haplogroup mtDNA sample was barcoded with unique DNA barcodes prior to pooling, decontamination and amplification. Complete decontamination was measured via sequencing the mixed libraries to 10,000× coverage and measuring heteroplasmy levels with and without MspJI decontamination. MspJI digestion removed 100% of expected heteroplasmy contaminants(red) suggesting it can decontaminate up to equimolar contamination events. Undigested pooled libraries were sequenced as a control (blue) and exhibited 35–65% heteroplasmy levels. These artificial heteroplasmies were produced by pooling a methylated mitochondrial Long Range PCR product from a different haplogroup into a non methylated product. This haplogroup is completely removed by the decontamination methods described.

Mentions: We measured decontamination by spiking in known amounts of DNA contaminant from a different mitochondrial haplogroup. Then, we treated these samples with the respective enzymes and deeply sequenced (10,000×) to measure the percent heteroplasmy of the sample at the haplogroup specific loci. A simple 1 hr digestion was able to remove equimolar contaminating DNA (Figure 5). This assay is limited in that it is only measuring contamination at 8 haplogroup specific loci.


Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment.

McKernan KJ, Spangler J, Zhang L, Tadigotla V, McLaughlin S, Warner J, Zare A, Boles RG - PLoS ONE (2014)

Decontamination effectiveness.To measure decontamination potential we mixed equimolar 5me-dCTP amplified mtDNA into non-methylated Target mtDNA. Methylated and non-methylated DNA were from mtDNA haplogroups differing in 8 loci. Each haplogroup mtDNA sample was barcoded with unique DNA barcodes prior to pooling, decontamination and amplification. Complete decontamination was measured via sequencing the mixed libraries to 10,000× coverage and measuring heteroplasmy levels with and without MspJI decontamination. MspJI digestion removed 100% of expected heteroplasmy contaminants(red) suggesting it can decontaminate up to equimolar contamination events. Undigested pooled libraries were sequenced as a control (blue) and exhibited 35–65% heteroplasmy levels. These artificial heteroplasmies were produced by pooling a methylated mitochondrial Long Range PCR product from a different haplogroup into a non methylated product. This haplogroup is completely removed by the decontamination methods described.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008621&req=5

pone-0096492-g005: Decontamination effectiveness.To measure decontamination potential we mixed equimolar 5me-dCTP amplified mtDNA into non-methylated Target mtDNA. Methylated and non-methylated DNA were from mtDNA haplogroups differing in 8 loci. Each haplogroup mtDNA sample was barcoded with unique DNA barcodes prior to pooling, decontamination and amplification. Complete decontamination was measured via sequencing the mixed libraries to 10,000× coverage and measuring heteroplasmy levels with and without MspJI decontamination. MspJI digestion removed 100% of expected heteroplasmy contaminants(red) suggesting it can decontaminate up to equimolar contamination events. Undigested pooled libraries were sequenced as a control (blue) and exhibited 35–65% heteroplasmy levels. These artificial heteroplasmies were produced by pooling a methylated mitochondrial Long Range PCR product from a different haplogroup into a non methylated product. This haplogroup is completely removed by the decontamination methods described.
Mentions: We measured decontamination by spiking in known amounts of DNA contaminant from a different mitochondrial haplogroup. Then, we treated these samples with the respective enzymes and deeply sequenced (10,000×) to measure the percent heteroplasmy of the sample at the haplogroup specific loci. A simple 1 hr digestion was able to remove equimolar contaminating DNA (Figure 5). This assay is limited in that it is only measuring contamination at 8 haplogroup specific loci.

Bottom Line: We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides.Use of this enzyme-and-nucleotide combination enables what we term a "DNA diode", where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays.Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment.

View Article: PubMed Central - PubMed

Affiliation: Courtagen Life Sciences, Woburn, Massachusetts, United States of America.

ABSTRACT
We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides. Use of this enzyme-and-nucleotide combination enables what we term a "DNA diode", where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays. Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment. These additional nucleotides enable a novel DNA decontamination technique that generates ephemeral and easy to decontaminate DNA.

Show MeSH