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Genome-wide mapping of DNA strand breaks.

Leduc F, Faucher D, Bikond Nkoma G, Grégoire MC, Arguin M, Wellinger RJ, Boissonneault G - PLoS ONE (2011)

Bottom Line: Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered.Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution.This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT
Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

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Enrichment of DNA sequences in the vicinity of a unique PciI restriction site present in the pcDNA3 plasmid.(a) Step-by-step workflow of the dDIP for the in vitro plasmid model (b) Qualitative determination of enrichment by multiplex PCR using decreasing copy numbers of plasmid DNA as indicated. Un, unbound; IP, immunoprecipitation.
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pone-0017353-g002: Enrichment of DNA sequences in the vicinity of a unique PciI restriction site present in the pcDNA3 plasmid.(a) Step-by-step workflow of the dDIP for the in vitro plasmid model (b) Qualitative determination of enrichment by multiplex PCR using decreasing copy numbers of plasmid DNA as indicated. Un, unbound; IP, immunoprecipitation.

Mentions: To demonstrate the specific capture and sensitivity of DNA strand breaks in vitro, we used a plasmid model (pcDNA3, Invitrogen, Burlington, ON, Canada) (Figure 2). First, pcDNA3-transformed DH5α E. coli were grown overnight and the plasmid was purified using a Qiagen Plasmid Midi Kit (Qiagen Inc., Mississauga, ON, Canada). Plasmid integrity was checked by 0.8% agarose gel electrophoresis (Promega corp., Madison, WI). Yield and purity was determined by spectrometry (Ultrospec 2100 pro, GE Healthcare, Piscataway, NJ). A unique double-stranded break was created using the endonuclease PciI (New England Biolabs, Ipswich, MA) by digesting one µg of plasmid with 10 U of enzyme in a final volume of 20 µl for 1 h at 37°C in the recommended buffer (NEB3). Thermal inactivation of PciI was obtained by raising the reaction temperature to 80°C for 20 min. This preparation is referred to as “digested” (Dig). Undigested pcDNA3 plasmid was used as negative control and termed “N”.


Genome-wide mapping of DNA strand breaks.

Leduc F, Faucher D, Bikond Nkoma G, Grégoire MC, Arguin M, Wellinger RJ, Boissonneault G - PLoS ONE (2011)

Enrichment of DNA sequences in the vicinity of a unique PciI restriction site present in the pcDNA3 plasmid.(a) Step-by-step workflow of the dDIP for the in vitro plasmid model (b) Qualitative determination of enrichment by multiplex PCR using decreasing copy numbers of plasmid DNA as indicated. Un, unbound; IP, immunoprecipitation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045442&req=5

pone-0017353-g002: Enrichment of DNA sequences in the vicinity of a unique PciI restriction site present in the pcDNA3 plasmid.(a) Step-by-step workflow of the dDIP for the in vitro plasmid model (b) Qualitative determination of enrichment by multiplex PCR using decreasing copy numbers of plasmid DNA as indicated. Un, unbound; IP, immunoprecipitation.
Mentions: To demonstrate the specific capture and sensitivity of DNA strand breaks in vitro, we used a plasmid model (pcDNA3, Invitrogen, Burlington, ON, Canada) (Figure 2). First, pcDNA3-transformed DH5α E. coli were grown overnight and the plasmid was purified using a Qiagen Plasmid Midi Kit (Qiagen Inc., Mississauga, ON, Canada). Plasmid integrity was checked by 0.8% agarose gel electrophoresis (Promega corp., Madison, WI). Yield and purity was determined by spectrometry (Ultrospec 2100 pro, GE Healthcare, Piscataway, NJ). A unique double-stranded break was created using the endonuclease PciI (New England Biolabs, Ipswich, MA) by digesting one µg of plasmid with 10 U of enzyme in a final volume of 20 µl for 1 h at 37°C in the recommended buffer (NEB3). Thermal inactivation of PciI was obtained by raising the reaction temperature to 80°C for 20 min. This preparation is referred to as “digested” (Dig). Undigested pcDNA3 plasmid was used as negative control and termed “N”.

Bottom Line: Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered.Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution.This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.

ABSTRACT
Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

Show MeSH
Related in: MedlinePlus