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Nuclease-assisted suppression of human DNA background in sepsis.

Song Y, Giske CG, Gille-Johnson P, Emanuelsson O, Lundeberg J, Gyarmati P - PLoS ONE (2014)

Bottom Line: Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons.With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome.According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

View Article: PubMed Central - PubMed

Affiliation: Royal Institute of Technology, Science for Life Laboratory, Stockholm, Sweden.

ABSTRACT
Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C0t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C0t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

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Related in: MedlinePlus

Standard curves and nuclease effect using amplicons to model excess human DNA.Human β-actin (a) and E. coli (b) primers provided the same R2 (>0.99) value and sensitivity (1–10 copies as the lower limit of detection) on amplicons in triplicate trials. ΔCt was 3.2 for human, 3.37 for E. coli. Error bars represent standard deviation.
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pone-0103610-g002: Standard curves and nuclease effect using amplicons to model excess human DNA.Human β-actin (a) and E. coli (b) primers provided the same R2 (>0.99) value and sensitivity (1–10 copies as the lower limit of detection) on amplicons in triplicate trials. ΔCt was 3.2 for human, 3.37 for E. coli. Error bars represent standard deviation.

Mentions: Ten-fold dilutions of human β-actin and E. coli were prepared from 100–109 copies (Figure 2). Standard curves were generated using the Bio-Rad CFX Manager software (v 1.6.541).


Nuclease-assisted suppression of human DNA background in sepsis.

Song Y, Giske CG, Gille-Johnson P, Emanuelsson O, Lundeberg J, Gyarmati P - PLoS ONE (2014)

Standard curves and nuclease effect using amplicons to model excess human DNA.Human β-actin (a) and E. coli (b) primers provided the same R2 (>0.99) value and sensitivity (1–10 copies as the lower limit of detection) on amplicons in triplicate trials. ΔCt was 3.2 for human, 3.37 for E. coli. Error bars represent standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116218&req=5

pone-0103610-g002: Standard curves and nuclease effect using amplicons to model excess human DNA.Human β-actin (a) and E. coli (b) primers provided the same R2 (>0.99) value and sensitivity (1–10 copies as the lower limit of detection) on amplicons in triplicate trials. ΔCt was 3.2 for human, 3.37 for E. coli. Error bars represent standard deviation.
Mentions: Ten-fold dilutions of human β-actin and E. coli were prepared from 100–109 copies (Figure 2). Standard curves were generated using the Bio-Rad CFX Manager software (v 1.6.541).

Bottom Line: Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons.With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome.According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

View Article: PubMed Central - PubMed

Affiliation: Royal Institute of Technology, Science for Life Laboratory, Stockholm, Sweden.

ABSTRACT
Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C0t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C0t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

Show MeSH
Related in: MedlinePlus