UTILITY OF A SWISH AND SPIT TECHNIQUE FOR THE COLLECTION OF BUCCAL CELLS FOR TAP HAPLOTYPE DETERMINATION

Objective: To demonstrate the utility of a ''swish and spit'' techniqu e as a nucleated cell source for transporter associated with antigen p rocessing (TAP) haplotype determination by molecular methods in large- scale clinical trials. Design: Twenty normal volunteers were recruited for this prospective feasibility study. From each subject, buccal or blood cells (or both) mere collected for use in TAP haplotype assignme nt by molecular methods and subjected to various storage conditions. M aterial and Methods: As an alternative to use of lymphocytes obtained by venipuncture, we developed a swish and spit technique for collectin g buccal cells for assigning TAP haplotype. For this technique, the su bject vigorously swishes isotonic saline in the mouth and expectorates it into a collection container. DNA is extracted from the buccal cell s by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. In addition, we compared DNA extracted from mouthwash specimens stored under various conditions to which a specimen might be exposed if mailed. Results: DNA extracted from buccal cells obtained by the swish and spit technique provided excellent templates for the p olymerase chain reaction (PCR), and subject acceptance of this method was universal. In all cases, assigning TAP haplotype by PCR amplificat ion of specific alleles with use of buccal or blood-derived specimens was successful. The integrity of the specimens was unaffected by stora ge at -20 degrees C, 4 degrees C, 25 degrees C, or 37 degrees C, and m e mere able to use the DNA from cells stored under any of these condit ions for TAP haplotyping. Conclusion: We conclude that DNA from buccal cells collected by the swish and spit technique for TAP haplotype ass ignment is an excellent substitute for DNA obtained from nucleated blo od cells, and the technique is useful for large-scale clinical studies that require DNA from subjects geographically distant from the resear ch site.