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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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Related in: MedlinePlus

Quantification by qPCR of immunoprecipitated DNA sequences flanking the PciI restriction site of the plasmid pcDNA3.Bars represent the average of two independent IP and error bars correspond to the standard deviation for qPCR measurements in triplicates. Dig+, PciI-digested pcDNA3 end-labeled with dATP, biotin-dATP and TdT; Dig-, PciI-digested pcDNA3 incubated with dATP and biotin-dATP without TdT; N+, intact pcDNA3 end-labeled with dATP, biotin-dATP and TdT.
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pone-0017353-g003: Quantification by qPCR of immunoprecipitated DNA sequences flanking the PciI restriction site of the plasmid pcDNA3.Bars represent the average of two independent IP and error bars correspond to the standard deviation for qPCR measurements in triplicates. Dig+, PciI-digested pcDNA3 end-labeled with dATP, biotin-dATP and TdT; Dig-, PciI-digested pcDNA3 incubated with dATP and biotin-dATP without TdT; N+, intact pcDNA3 end-labeled with dATP, biotin-dATP and TdT.

Mentions: To demonstrate the specificity and sensitivity of the DNA strand breaks capture, we first used a plasmid as a model representing a simple defined hotspot (Figure 2 and 3). To simulate a DSB, pcDNA3 was digested at a unique restriction site by the endonuclease PciI. The DNA ends were labeled by the incorporation of biotin-modified nucleotides (dUTP or dATP) at 3′OH termini by the TdT. The labeled DNA was further digested in fragments ranging from 4 to 720 bp by the endonuclease NlaIII which will generate probes of suitable size when applied to a genomic context. We first verified the presence of specific fragments α and β, flanking the PciI restriction site and also of the fragment ε, as an internal negative control, using multiplex PCR amplification as illustrated in Figure 2. The results show specific capture of fragments α and β, whereas the fragment ε is not present in the IP fraction and remains in the unbound fraction. Specific capture was successfully achieved with as low as 10 copies of pcDNA3 demonstrating the sensitivity of the capture method.


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)

Quantification by qPCR of immunoprecipitated DNA sequences flanking the PciI restriction site of the plasmid pcDNA3.Bars represent the average of two independent IP and error bars correspond to the standard deviation for qPCR measurements in triplicates. Dig+, PciI-digested pcDNA3 end-labeled with dATP, biotin-dATP and TdT; Dig-, PciI-digested pcDNA3 incubated with dATP and biotin-dATP without TdT; N+, intact pcDNA3 end-labeled with dATP, biotin-dATP and TdT.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017353-g003: Quantification by qPCR of immunoprecipitated DNA sequences flanking the PciI restriction site of the plasmid pcDNA3.Bars represent the average of two independent IP and error bars correspond to the standard deviation for qPCR measurements in triplicates. Dig+, PciI-digested pcDNA3 end-labeled with dATP, biotin-dATP and TdT; Dig-, PciI-digested pcDNA3 incubated with dATP and biotin-dATP without TdT; N+, intact pcDNA3 end-labeled with dATP, biotin-dATP and TdT.
Mentions: To demonstrate the specificity and sensitivity of the DNA strand breaks capture, we first used a plasmid as a model representing a simple defined hotspot (Figure 2 and 3). To simulate a DSB, pcDNA3 was digested at a unique restriction site by the endonuclease PciI. The DNA ends were labeled by the incorporation of biotin-modified nucleotides (dUTP or dATP) at 3′OH termini by the TdT. The labeled DNA was further digested in fragments ranging from 4 to 720 bp by the endonuclease NlaIII which will generate probes of suitable size when applied to a genomic context. We first verified the presence of specific fragments α and β, flanking the PciI restriction site and also of the fragment ε, as an internal negative control, using multiplex PCR amplification as illustrated in Figure 2. The results show specific capture of fragments α and β, whereas the fragment ε is not present in the IP fraction and remains in the unbound fraction. Specific capture was successfully achieved with as low as 10 copies of pcDNA3 demonstrating the sensitivity of the capture method.

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