UNIQUE PANCREATIC-CARCINOMA CDNA FOUND VIA SUBTRACTED LIBRARIES WITH LIMITED NORMAL TISSUE AND TUMOR FROM THE SAME PATIENT

We have approached the problem of isolating clones unique to specific malignancies such as T-cell acute lymphoblastic leukemia (ALL) and pan creatic adenocarcinoma by using subtractive hybridization techniques. Our initial studies involved using normal donor tissue (i. e, normal b lood donors for ALL and cadaver renal transplant donors for normal pan creatic tissue) and cultured malignant cell lines. It occurred to us t hat normal pancreatic tissue from the same patient source as that of t he malignant tissue might subtract out normal sequences more readily a nd enrich clones unique to pancreatic adenocarcinoma because of patien t/donor identity. Using such a method meant that the amounts of tissue for overcoming this obstacle. We constructed independent UNI-ZAP-XR c DNA libraries (normal and malignant) and used them to amplify either t he normal or malignant cDNA prior to subtractive hybridization. We the n obtained rescued single stranded cDNA from the malignant ZAP library . The RNA which was not hybridized was isolated. The process was repea ted and a double subtraction was effected. The residual non-hybridized RNA was used as a template for first and second strand synthesis. Aft er the EcoRI adaptors were ligated to the double stranded cDNA it was cloned into Lambda ZAP II arms to form a double subtracted malignant c DNA library. A subtracted probe was prepared from the double subtracte d cDNA library. Single stranded cDNA was rescued, double stranded plas mid was made, the plasmid DNA was digested with EcoRI, the digested DN A was run on a 1% SeaPlaque gel, and the insert cDNA was recovered usi ng Ultra-Free MC and Ultra-Free Probind filters. The subtracted malign ant cDNA library was probed with the subtracted probe and with normal cDNA (obtained from the normal ZAP library) and those plaques which we re positive per the subtracted probe and negative per the normal cDNA were isolated; their cDNA inserts are being further characterized.