Departmental Cores (DC)

Human Gene Linkage and Genetics Assessment

Annenberg Building, 17th Floor, Room 17-30
One Gustave L. Levy Place
New York, NY 10029

Contact Persons

Director: Robert J. Desnick, M.D., Ph.D.
E-mail: robert.desnick@mssm.edu
Tel: (212) 241-8662 or ext 48662

Co-Director: Bruce Gelb, M.D.
E-mail: bruce.gelb@mssm.edu
Tel: (212) 241-8662 or ext 48662

This Core provides expertise for the mapping of human genes. The first task in gene discovery is to map the genes responsible for traits of interest. Both traits with simple Mendelian modes of inheritance and traits with complex patterns of inheritance can be studied. The Core facility can determine genotypes for highly polymorphic markers that systematically cover the entire genome for linkage and association analysis. The Core is equipped with a Beckman Biomek robotic, Perkin-Elmer 9600. Using this equipment, the genotype data will be determined by sizing radiolabeled PCR products by electrophoresis or fluoroscently-labeled PCR products on the ABI sequencer. The absolute sizes of the products will be determined using internal standards added to each sample. Automation (Biomek) is used to setup all PCR reactions to increase reproducibility.

The Core facility will initially have the capacity to determine 1,000 genotypes per day. A typical screen of the genome requires 380 genotypes per individual which can be done with approximately 10-15 ml of whole blood (60-90 micrograms of DNA). Genotype data is collected in a computer-readable form for analysis. Expertise in genotype data analysis is available in the Core. Subsequent to the detection of linkage, the Core can determine genotypes for additional markers to determine the minimum obligate region which must contain the gene of interest.

Expert assistance is available for study design, sample procurement, establishment of long-term lymphoid lines and linkage data analysis. The facility also provides expertise to gain access to current genomics information which is critical for all positional cloning efforts. In addition, the Core offers molecular genetic technology to Medical School investigators engaged in fundamental or applied research on the genetic basis of human diseases. Gene mapping, molecular diagnostics, and establishment of human cell lines are the major technical services rendered. In addition, members of the Core staff are available to provide assistance in their areas of expertise on a consultative or collaborative basis.

MOLECULAR DIAGNOSTICS for human diseases with a genetic component provided by the Core includes prenatal, presymptomatic and carrier DNA-based tests for diseases such as cystic fibrosis, sickle cell disease, Tay-Sachs disease, al-antitrypsin deficiency, Fabry disease, Gaucher disease and the inherited porphyrias. The diagnoses are based on the identification of common mutations by PCR techniques (detected with restriction enzymes, mutation-specific oligonucleotides, or DNA sequencing), and/or family studies using gene-specific or closely-linked RFLPS. Additional selected diagnostic tests will be incorporated into the Core's repertoire as new methods of molecular diagnosis become available or if sufficient interest is generated.

The HUMAN GENE MAPPING facility provides somatic cell hybrid panels and/or DNA isolated from these panels for the chromosomal mapping of human genes. Currently, several human/rodent somatic cell hybrid panels containing single or multiple human chromosomes on mouse or hamster backgrounds are available. These panels can be used to map human genes to specific chromosomes by protein and/or DNA analysis techniques. PCR analyses can be performed on DNA from the hybrid panels for chromosome assignment. In addition, filters can be prepared containing various restriction digests of each hybrid panel for subsequent Southern hybridization analysis with species-specific CDNA and/or oligonucleotide probes. Hybrid cells containing specific gene rearrangements also are available for many human chromosomes and are useful for regional chromosomal assignments

The HUMAN CELL CULTURE facility serves investigators requiring established lines of cultured human fibroblasts from skin biopsies and of cultured human lymphoid cells from venous blood samples. Fibroblasts are established at greater than 98% success rate. Permanent lymphoblast cell lines are established with greater than 90% success rate; these transformed cells are particularly useful for molecular biology experiments since large quantities (> l08 cells) can be grown for DNA and/or RNA isolation. The Core staff will also maintain, freeze-down, and store the various lines used by investigators. Each frozen cell line is stored in two separate liquid nitrogen containers to insure independent safe keeping.