REAMPLIFICATION OF DIFFERENTIALLY EXPRESS ED FRAGMENTS FROM HUMAN HEAD AND NECK SQUAMOUS-CELL CARCINOMA-CELLS FOR SEQUENCING REQUIRING NO MOLECULAR-CLONING

Background: mRNA expression of healthy and malignant cells can be comp ared to each other by employing the ''differential display'' (DD) tech nique. Most studies describe sequence analysis of differentially expre ssed fragments after reamplification by a second round of PCR and subs equent molecular cloning to gain a sufficient amount of DNA for sequen cing. The aim of this study was to show whether a sufficient amount of differentially expressed mRNA of squamous cell carcinoma cells of the head and neck region can be generated by PCR alone without cloning st eps. Material and Methods: mRNA isolated from cultivated keratinocytes and squamous cell carcinoma cells was reverse transcribed into cDNA w hich was amplified with PCR. Differentially expressed fragments detect ed after gel electrophoresis were isolated from the gel and reamplifie d in a second PCR. The resulting cDNA amounts of the second PCR were s uitable for cloning but not for direct sequencing. A third round of PC R with the undiluted final product of the second PCR as template regul arly failed. Dilutions of the second PCR products between 1:10 and 1:1 0(10) were prepared. The third round of PCR was carried out with these various template concentrations. Results: A sufficient amount of diff erentially expressed fragments for sequencing procedures resulted when dilutions of the second PCR products ranging from 1:10(2) to 1:10(7) were used as templates in the third round of PCR. Conclusion: Modifica tions of PCR parameters provide high DNA copy numbers of differentiall y expressed mRNA fragments from squamous cell carcinoma cells of the u pper aerodigestive tract in amounts that are needed for sequence analy sis. This may make it possible to avoid labor-intensive cloning proced ures requiring high safety standards.