Dr. Jacques R. Fresco
Department of Molecular Biology
Princeton University
Princeton, NJ 08544
Phone: 609-258-3927
Fax: 609-258-6730
Email: jrfresco@princeton.edu
Projects are approved by an IRB located at: Department of Molecular Biology, Princeton University.
The approving IRB does not operate under a Multiple Project Assurance (MPA) recognized by DOE or by the Department of Health and Human Services (HHS).
Number of Human Subjects Projects reported: 1
Project Identifier: PU-96-DE-FG02-96ER62202
Project Title:
Development of Affinity Technology for Isolating Individual Human Chromosomes by Third Strand Binding
Principal Investigator:
Dr. Jacques R. Fresco
Principal Investigator's Institution: Department of Molecular Biology
Project started in: 1996
Project Funding Information:
Project received funding in Fiscal Year 1996.
Project used human subjects in Fiscal Year 1996.
Funding Sources:
Blood samples taken at the Princeton University Infirmary were provided by the principal investigator and the Ph.D. researcher of the grant, all at no cost
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: November 01, 1996
Number of Human Subjects who participated in this project/protocol during
FY 1996 (10/1/95 - 9/30/96): 2
Type of Human Subjects Involvement:
A. OBJECTIVE
The aim of this project is to exploit "third-strand" nucleic acid binding, which involves non-denaturing conditions, to target duplexes for the purpose of developing new technologies for chromosome identification, sorting, and bulk isolation. The respective technologies, triplex in situ hybridization (TISH), triplex univariate flow sorting, and triplex affinity chromosome purification, depend on the design of nucleic acid third-strands to bind with great specificity and affinity via triple helix formation to repetitive double helical centromeric alpha satellite DNA target sequences unique to individual human chromosomes.
A single copy of the alphoid DNA target sequence is present within each tandemly repeated higher order alpha-satellite DNA array. These arrays, are, in turn, located in an anatomically unique chromosomal region, the centromere, where they are likely to be more accessible to third-strand binding than endogenous hetero-and euchromatin. Moreover, target sequences for third-strand binding have a high probability of being chromosome-specific since alphoid DNAs differ with respect to their degree of sequence relatedness.
B. METHODOLOGY
We outline below the generic approach that should allow third-strand probes to bind to any human metaphase chromosome in situ and in suspension.
1. Selection of a centromeric alphoid DNA target sequence that is unique to a particular human chromosome and present in multiple copies. A database search has confirmed the presence of unique alpha-satellite target sequences in 21 of the 24 human chromosomes.
2. Design of oligodeoxynucleotide third-strand affinity probes for binding to alphoid DNA target sequences.
3. Determination that the third-strand affinity probes can bind specifically to their alphoid DNA target sequences in recombinant DNA molecules under buffer conditions optimal for chromosome hybridization.
4. Confirmation by Triplex In Situ Hybridization (TISH) of third-strand binding to duplex alphoid DNA targets on metaphase chromosome spreads from cultured human lymphocytes.
5. Development of Triplex Suspension Hybridization (TSH), and if necessary, gentle extraction procedures to remove centromere-bound proteins.
6. Determination of ability of biotinylated third strands to affinity capture the correct metaphase chromosome from a suspension using different streptavidin-based approaches.
7. Determination of conditions for bulk isolation and purification of the correct metaphase chromosome by third strand affinity capture.
C. NOT APPLICABLE
D. INVOLVEMENT OF HUMAN SUBJECTS
No statistical sample data collection is involved. A few healthy human blood donors from our own research group donate 10 ml of blood, drawn at the Princeton University Infirmary under standard sterile conditions. Aliquots of this blood are cultured under standard sterile conditions in order to obtain metaphase and interphase lymphocytes for cytogenetic analysis. There are no risks to human donors, and all subsequent studies are performed in vitro. No public references to sources of donors are planned; and blood samples are volunteered by the researchers themselves.