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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.

Show MeSH
Quantitative PCR of digested and undigested Déjà vu libraries.120 minute digestion of AbaSI at 25°C on methylated DNA and hydroxymethylated DNA. A 100 fold reduction in background hydroxymethylated DNA is obtained with a 2 hr 25°C digestion with 0.3Units of Enzyme.
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pone-0096492-g003: Quantitative PCR of digested and undigested Déjà vu libraries.120 minute digestion of AbaSI at 25°C on methylated DNA and hydroxymethylated DNA. A 100 fold reduction in background hydroxymethylated DNA is obtained with a 2 hr 25°C digestion with 0.3Units of Enzyme.

Mentions: After the first LR-PCR and prior to the second Nextera PCR we use AbaSI to digest contaminants as this enzyme only digests 5-hmeC, leaving 5-meC or cytosine intact. In this case, AbaSI will only digest PCR products that contaminate the pre-Nextera sample from the post-secondary PCR process (Figure 3). The second PCR usually contains universal sequencing primers producing small products (700 bp) desired by the limitations of current sequencers. These smaller PCR products can hyper-amplify due to cold PCR or other selective amplification biases and as a result can be over represented. Hyper-amplification of contaminants in PCR a risk in a clinical laboratory testing for heteroplasmy [36].


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)

Quantitative PCR of digested and undigested Déjà vu libraries.120 minute digestion of AbaSI at 25°C on methylated DNA and hydroxymethylated DNA. A 100 fold reduction in background hydroxymethylated DNA is obtained with a 2 hr 25°C digestion with 0.3Units of Enzyme.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0096492-g003: Quantitative PCR of digested and undigested Déjà vu libraries.120 minute digestion of AbaSI at 25°C on methylated DNA and hydroxymethylated DNA. A 100 fold reduction in background hydroxymethylated DNA is obtained with a 2 hr 25°C digestion with 0.3Units of Enzyme.
Mentions: After the first LR-PCR and prior to the second Nextera PCR we use AbaSI to digest contaminants as this enzyme only digests 5-hmeC, leaving 5-meC or cytosine intact. In this case, AbaSI will only digest PCR products that contaminate the pre-Nextera sample from the post-secondary PCR process (Figure 3). The second PCR usually contains universal sequencing primers producing small products (700 bp) desired by the limitations of current sequencers. These smaller PCR products can hyper-amplify due to cold PCR or other selective amplification biases and as a result can be over represented. Hyper-amplification of contaminants in PCR a risk in a clinical laboratory testing for heteroplasmy [36].

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