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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

Multiplex PCR analysis of the captured DNA sequences in the vicinity of a unique PciI restriction site on the plasmid pcDNA3 transformed in DH5α E. coli and embedded in 1% agarose plugs.
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pone-0017353-g004: Multiplex PCR analysis of the captured DNA sequences in the vicinity of a unique PciI restriction site on the plasmid pcDNA3 transformed in DH5α E. coli and embedded in 1% agarose plugs.

Mentions: As a first application of the dDIP technique in a genomic context, we applied the same strategy within a prokaryotic genome background. Overnight culture of E. coli DH5α transformed with pcDNA3 was embedded in 1% low melting agarose to minimize DNA fragmentation due to handling before labeling DNA ends. After lysis and protein digestion, DNA was digested in agarose plugs using PciI and labeled by TUNEL. After β-agarase digestion of the plugs, total DNA (genomic and plasmidic) was precipitated and further digested by NlaIII. Presence of the specific plasmid fragments was assessed by multiplex PCR amplification (see Figure 4). As for the experiment described above with the purified plasmid, specific capture of fragments α and β with limited background from fragment ε was obtained was again demonstrated.


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)

Multiplex PCR analysis of the captured DNA sequences in the vicinity of a unique PciI restriction site on the plasmid pcDNA3 transformed in DH5α E. coli and embedded in 1% agarose plugs.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017353-g004: Multiplex PCR analysis of the captured DNA sequences in the vicinity of a unique PciI restriction site on the plasmid pcDNA3 transformed in DH5α E. coli and embedded in 1% agarose plugs.
Mentions: As a first application of the dDIP technique in a genomic context, we applied the same strategy within a prokaryotic genome background. Overnight culture of E. coli DH5α transformed with pcDNA3 was embedded in 1% low melting agarose to minimize DNA fragmentation due to handling before labeling DNA ends. After lysis and protein digestion, DNA was digested in agarose plugs using PciI and labeled by TUNEL. After β-agarase digestion of the plugs, total DNA (genomic and plasmidic) was precipitated and further digested by NlaIII. Presence of the specific plasmid fragments was assessed by multiplex PCR amplification (see Figure 4). As for the experiment described above with the purified plasmid, specific capture of fragments α and β with limited background from fragment ε was obtained was again demonstrated.

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