Brind'Amour

Tuesday, July 5, 2011, 9:00 am. Room 203, Graduate Student Centre

“Flow Cytometry Analysis and Sorting of Chromosomes Following Hybridization with Fluorescent Probes that Target Specific DNA Repeat Sequences”

 

EXAMINING COMMITTEE

Chair:
    Dr. Leslie D. Burtnick (Chemistry)

Supervisory Committee:
    Dr. Peter Lansdorp, Research Supervisor (Medical Genetics)
    Dr. Carolyn Brown (Medical Genetics)
    Dr. Louis Lefebvre (Medical Genetics)

University Examiners:
    Dr. Andrew Weng (Pathology and Laboratory Medicine)
    Dr. Fabio Rossi (Medical Genetics)

External Examiner:
    Dr. M. Katharine Rudd
    Department of Human Genetics
    Emory University School of Medicine
    Atlanta, Georgia
    USA


SUPERVISORY COMMITTEE
Dr. Peter Lansdorp, supervisor
Dr. Carolyn Brown (Medical Genetics)
Dr. Louis Lefebvre (Medical Genetics)
Dr. Tom Grigliatti (Zoology)



ABSTRACT
Traditional cytogenetic approaches allow analysis of the chromosomal composition (karyotype) of mitotic cells fixed on slides cells by microscopy. The combination of karyotyping and Fluorescence In Situ Hybridization (FISH) enables the detection of specific target sequences on individual chromosomes. Disadvantages are that traditional cytogenetic approaches are very labor and time consuming and that chromosome specific information from only a few dozen cells has poor statistical power. An alternative is flow karyotyping, a method to analyze chromosomes in suspension by flow cytometry. For flow karyotyping, the DNA composition of specific chromosomes in suspension is measured based on the DNA-specific dyes Hoechst 33258 (HO) and Chromomycin A3 (CA3). My thesis work has focused on the development of a new method to analyze and sort chromosomes using FISH with labeled peptide nucleic acid (PNA) probes on chromosomes in suspension. I found that, following FISH, flow karyotyping can be used to detect and quantify repetitive DNA sequences within individual chromosomes.
Using chromosome flow FISH (CFF), chromosomes isolated from cells of various species were hybridized to PNA probes and analyzed by flow cytometry. CFF was used to detect a variety of repeats; interstitial telomeric sequences in Chinese Hamster chromosomes, major satellite in mouse chromosomes and L1.84 alpha satellite repeats in human chromosomes. Quantitative measurements of repeat length by CFF were validated by comparison with measurements obtained using Q-FISH. We found that parental homologs of human chromosome 18 with different L1.84 satellite repeat length could be purified using CFF and Fluorescence Activated Cell Sorting (FACS). Illumina short read sequencing of libraries built from these purified chromosomes enabled us to determine, with a high resolution, the allelic phasing of each homolog over the entire chromosome 18. Finally, CFF was modified to study sister chromatids separately. Using a cell model with inducible separation of sister chromatids, flow karyograms were generated. Using chromosome orientation FISH (CO-FISH) in suspension, we could identify sister chromatids according to the presence of DNA template strands. We anticipate that this approach will allow the purification of sister chromatids to study epigenetic differences between sister chromatids defined on the basis of DNA template strands.



PUBLICATIONS
Brind'Amour, J. and Lansdorp, P. M. (2011). "Analysis of repetitive DNA in chromosomes by flow cytometry." Nat Meth 8(6): 484-486.

Brind'Amour, J. and Lansdorp P. M. (2011). Peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) on chromosomes in suspension for analysis of repetitive DNA by flow cytometry. Protocol Exchange, Nature Publishing Group.

Shah G.M., K.-K. F., Montoni A., Shah R.G., Brind'Amour J., Vodenicharov M.D. and Affar E.B. (2011). “Approaches to Detect PARP-1 Activation In vivo, In situ, and In vitro” in Poly(ADP-ribose) Polymerase. A. Tulin, Humana Press. (In Press)


PRESENTATIONS
Julie Brind’Amour, Lindsey Laycock, Gary de Jong and Peter M. Lansdorp. Chromosome specific telomere repeats measurements by flow cytometry. Canadian symposium on telomeres and telomerase, Hamilton, May 2010. (Oral presentation)

Julie Brind’Amour, Lindsey Laycock, Gary de Jong and Peter M. Lansdorp. Measurements of repetitive DNA in Chromosomes by Flow cytometry. International Society for Analytical Cytology CYTO 2010 meeting, Seattle, May 2010. (Poster)

Julie Brind’Amour, Lindsey Laycock, Gary de Jong and Peter M. Lansdorp. Flow cytometry to quantify telomere repeats on specific chromosomes in suspension. Cold Spring Harbor Meeting on Telomeres and Telomerase, Cold Spring Harbor, May 2009 (Poster)

Julie Brind’Amour, Lindsey Laycock, Elizabeth Chavez and Peter M. Lansdorp. Fluorescent In Situ Hybridization on Suspension Chromosomes to Analyze Sequence Specific Signal by Flow Cytometry. Medical Genetics Research Day, Vancouver, November 2008 (Poster)

Julie Brind’Amour, Judith Banat, Peggy Olive and Peter M. Lansdorp. Measurement of DNA damage at the telomeres by Comet-FISH. Canadian symposium on telomeres and telomerase, Calgary, 2006 (Poster)

Julie Brind’Amour, Rashmi G. Shah and Girish M. Shah. Role of poly(ADP-ribose) polymerase in the early responses to ultraviolet B radiation in the epidermis of SKH-1 hairless mice. PARP meeting 2005: Bench to bedside, New Castle upon Tyne (UK), 2005. (Poster)


BIOGRAPHICAL NOTES
Born: November 8, 1979
Academic Studies: B. Sc. Université Laval, 2003
M. Sc. Université Laval, 2005


GRADUATE STUDIES
Field of Study: Molecular cytogenetics
Courses:
MEDG 520: Advanced Human Molecular Genetics. Dr. A. Brooks-Wilson
MEDG 530: Advanced Human Genetics. Dr. J. Friedman

 

AWARDS
2010: Canadian Symposium on Telomeres and Telomerase: Oral presentation prize
2006-2010: University of British Columbia PhD tuition Award