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Tracking chromatin states using controlled DNase I treatment and real-time PCR

Abstract

A novel approach to DNase I-sensitivity analysis was applied to examining genes of the spermatogenic pathway, reflective of the substantial morphological and genomic changes that occur during this program of differentiation. A new real-time PCR-based strategy that considers the nuances of response to nuclease treatment was used to assess the nuclease susceptibility through differentiation. Data analysis was automated with the K-Lab PCR algorithm, facilitating the rapid analysis of multiple samples while eliminating the subjectivity usually associated with Ct analyses. The utility of this assay and analytical paradigm as applied to nuclease-sensitivity mapping is presented.

Abbreviations

Actb:

β-actin

BSA:

bovine serum albumin

Ct :

cycle crossover threshold

DNase I:

deoxyribonuclease I

EDTA:

ethylenediamine tetraacetic acid

Emax :

maximum efficiency of PCR reaction

FSB:

frozen storage buffer

Hbb:

β-globin

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic

NP40:

Nonidet-P40

PBS:

phosphate buffered saline

PCR:

polymerase chain reaction

Prm:

protamine

Tnp2:

transition protein 2

RPMI-1640 Medium:

Liquid Media

Tn :

concentration of template at cycle n

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Correspondence to Stephen A. Krawetz.

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Martins, R.P., Platts, A.E. & Krawetz, S.A. Tracking chromatin states using controlled DNase I treatment and real-time PCR. Cell Mol Biol Lett 12, 545–555 (2007). https://doi.org/10.2478/s11658-007-0024-z

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Key words

  • DNase I-sensitivity
  • Differentiation
  • Spermatogenesis
  • Method