Techniques

We use a wide array of techniques and methodologies to accomplish our research goals. Some of them are listed below:

Molecular Biology

We use several molecular biology techniques including restriction-based molecular cloning, PCR, site-directed mutagenesis, DNA/RNA isolation from human cells, cDNA synthesis, real time PCR etc…

Protein expression and purification

We express proteins/complexes recombinantly in various systems including E. coli, baculovirus-infected insect cells, and cultured human cells for use in biochemical and X-ray crystallographic experiments.

Biochemical assays

We employ a wide-range of biochemical assays for determining binding constants, enzymatic activity etc. of various telomeric and telomerase complexes. Some examples are shown below.

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A native gel-shift assay showing that POT1 binds only DNA of telomeric sequence and not RNA.
Direct telomerase activity assay. Telomerase expressed in HeLa is purified and assayed for telomeric DNA extension. Shown is how WT TPP1 increases telomerase processivity (ability to synthesize bands running higher on the gel), whereas some mutants (in red) are greatly defective.
Direct telomerase activity assay. Telomerase expressed in HeLa is purified and assayed for telomeric DNA extension. Shown is how WT TPP1 increases telomerase processivity (ability to synthesize bands running higher on the gel), whereas some mutants (in red) are greatly defective.

X-ray Crystallography

We use X-ray crystallography as a major tool for determining the molecular bases of the various biological and biochemical phenomena that we investigate.

Parts of the X-ray crystal structure of the telomeric protein POT1 with an oligonucleotide containing one ribonucleotide (rU4) showing how POT1 binds DNA but not RNA
Parts of the X-ray crystal structure of the telomeric protein POT1 with an oligonucleotide containing one ribonucleotide (rU4) showing how POT1 binds DNA but not RNA

Cell biology

We use a wide range of cell biological assays ranging from immunofluorescence (IF) and fluorescence in situ hybridization (FISH) imaging of fixed human cells in culture, telomere length analysis, immunoprecipitation (IP), ChIP etc… We also use state-of-the-art techniques to engineer cell lines expressing genetic constructs of our interests in a stable and controlled fashion. Most recently we have successfully used CRISPR-Cas9 technology to introduce both knock out and knock in (single-site changes) mutations in telomeric genes in cultured human cells. Some examples of methods used in the lab are shown below.

Yellow in merge indicates telomerase is recruited to chromosome ends. Figure shows how telomerase is recruited in WT TPP1 cells (see yellow spots in merge), whereas mutant cells show complete lack of telomerase recruitment (red and green spots are separated in space).
Yellow in merge indicates telomerase is recruited to chromosome ends. Figure shows how telomerase is recruited in WT TPP1 cells (see yellow spots in merge), whereas mutant cells show complete lack of telomerase recruitment (red and green spots are separated in space).
Telomeres isolated from HeLa are probed by Southern Blot. Observe how WT TPP1 stimulates telomere lengthening (smears march up the gel), whereas mutant TPP1 does not.
Telomeres isolated from HeLa are probed by Southern Blot. Observe how WT TPP1 stimulates telomere lengthening (smears march up the gel), whereas mutant TPP1 does not.

CRISPR-Cas9 technology

We have successfully optimized both knock out and knock in of genes in cultured cells (like HEK 293T and HeLa) using CRISPR-Cas9 technology. Knock in changes include point mutations as well as insertion of epitope tags such as FLAG tags.

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