Objective: To demonstrate the utility of a "swish and spit" technique as a nucleated cell source for transporter associated with antigen processing (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) were collected for use in TAP haplotype assignment by molecular methods and subjected to various storage conditions.
Material and methods: As an alternative to use of lymphocytes obtained by venipuncture, we developed a swish and spit technique for collecting buccal cells for assigning TAP haplotype. For this technique, the subject vigorously swishes isotonic saline in the mouth and expectorates it into a collection container. DNA is extracted from the buccal cells 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 polymerase chain reaction (PCR), and subject acceptance of this method was universal. In all cases, assigning TAP haplotype by PCR amplification of specific alleles with use of buccal or blood-derived specimens was successful. The integrity of the specimens was unaffected by storage at -20 degrees C, 4 degrees C, 25 degrees C, or 37 degrees C, and we were able to use the DNA from cells stored under any of these conditions for TAP haplotying.
Conclusion: We conclude that DNA from buccal cells collected by the swish and spit technique for TAP haplotype assignment is an excellent substitute for DNA obtained from nucleated blood cells, and the technique is useful for large-scale clinical studies that require DNA from subjects geographically distant from the research site.