Tissue to Gene (S6)

A major goal of human genetics is to develop a complete map of the human genome. A high-resolution map of the genome is considered to be essential for diagnosis of genetic diseases and development of cures of these diseases by gene therapy. Studies on a specific DNA locus in a mammal usually begin with the isolation of genomic DNA from a tissue. The DNA is quantified, subjected to restriction nuclease digestion, and the fragments separated by gel electrophoresis. The DNA fragments in the gel are then transferred to a special membrane by the method of Southern and the specific DNA fragment under investigation is detected by hybridization analysis. Your students will perform each of these steps with this program series of five 3-hour laboratory sessions. The starting material for the series is calf thymus tissue. Students isolate DNA from purified thymus nuclei and then quantify it with a novel solid phase DNA assay. The DNA is digested with EcoR1, electrophoresed, and the separated fragments transferred to a nylon membrane. The DNA fragment containing the G+C-rich cow satellite is then detected by hybridization using a biotin-labeled probe made from a plasmid that contains the cow satellite sequence. Students also determine if the satellite sequence is found in sheep DNA. Microscopes, ethyl alcohol, a water bath incubator that will maintain a temperature of 60-65°C, and a centrifuge that can be operated at a force of at least 3000 x g are needed for the program.

A typical laboratory schedule for this program is given below where each lab session is approximately 3 hours.

Lab Session 1

Students isolate nuclei from thymus tissue and isolate thymus DNA from the nuclei by a unique procedure that yields high quality cellular DNA without the use of toxic organic solvents.

Lab Session 2

The purified DNA is quantified by a solid phase assay, digested with the restriction endonuclease EcoR1, and then electrophoresed on agarose gels.

Lab Sessions 3-5

The DNA fragments in the gels are transferred to nylon membranes using the transfer devices provided with the program.

The fragment containing the satellite sequence is then detected by hybridization using a biotin-labeled probe prepared from a plasmid containing the satellite sequence.