Archive for the ‘Stuart Lindsay’s Lab’ Category

Developing DNA tiles for oligonucleotide hybridization assay with higher accuracy and efficiency (pdf)

Saturday, February 27th, 2010

Developing DNA tiles for oligonucleotide hybridization assay with higher accuracy and efficiency

ABSTRACT

We demonstrate the versatility of a DNA tile system for oligonucleotide hybridization assay and explored the detection limit of the probe tiles for DNA targets of varied lengths.

Chromatin Stability at Low Concentration Depends on Histone Octamer Saturation Levels (pdf)

Saturday, February 27th, 2010

Chromatin Stability at Low Concentration Depends on Histone Octamer Saturation Levels

ABSTRACT

Studies on the stability of nucleosome core particles as a function of concentration have indicated a lower limit of ~5 ng/mL, below which the complexes start to spontaneously destabilize. Until recently little information was available on the effect of low concentration on chromatin. Using the well-characterized array of tandemly repeated 5S rDNA reconstituted into chromatin, we have investigated the effect of dilution. In this study, we demonstrate that the stability of saturated nucleosomal arrays and that of nucleosome core particles are within the same order of magnitude, and no significant loss of histones is monitored down to a concentration of 2.5 ng/mL. We observed that levels of subsaturation of the nucleosomal arrays were directly correlated with an increased sensitivity to histone loss, suggesting a shielding effect. The loss of histones from our linear nucleosomal arrays was shown not to be random, with a significant likelihood to occur at the end of the template than toward the center. This observation indicates that centrally located nucleosomes are more stable than those close to the end of the DNA templates. Itis important to take this information into account for the proper design of experiments pertaining to histone composition and the folding ability of chromatin samples.

Recognition Imaging of Acetylated Chromatin Using a DNA Aptamer (pdf)

Saturday, February 27th, 2010

Recognition Imaging of Acetylated Chromatin Using a DNA Aptamer

ABSTRACT

Histone acetylation plays an important role in the regulation of gene expression. A DNA aptamer generated by in vitro selection to be highly specific for histone H4 protein acetylated at lysine 16 was used as a recognition element for atomic force microscopy-based recognition imaging of synthetic nucleosomal arrays with precisely controlled acetylation. The aptamer proved to be reasonably specific at recognizing acetylated histones, with recognition efficiencies of 60% on-target and 12% off-target. Though this selectivity is much poorer than the >2000:1 equilibrium specificity of the aptamer, it is a large improvement on the performance of a ChIP-quality antibody, which is not selective at all in this application, and it should permit high-fidelity recognition with repeated imaging. The ability to image the precise location of posttranslational modifications may permit nanometer-scale investigation of their effect on chromatin structure.

The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure (pdf)

Saturday, February 27th, 2010

The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure

ABSTRACT

Eukaryotic DNA is packaged into chromatin, which regulates genome activities such as telomere maintenance. This study focuses on the interactions of a myb/SANT DNA-binding domain from the telomere-binding protein, TRF2, with reconstituted telomeric nucleosomal array fibers. Biophysical characteristics of the factor-bound nucleosomal arrays were determined by analytical agarose gel electrophoresis (AAGE) and single molecules were visualized by atomic force microscopy (AFM). (more…)