A high frequency of sequence alterations is due to formalin fixation of archival specimens

Genomic analysis of archival tissues fixed in formalin Is of fundamental im portance in biomedical research, and numerous studies have used such materi al. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overco me such problems, Here we report the results from a controlled study, perfo rmed in parallel on frozen and formalin-fixed material, where a high freque ncy of nonreproducible sequence alterations was detected with the use of fo rmalin-fixed tissues, Defined numbers of well-characterized tumor cells wer e amplified and anal);zed by direct DNA sequencing. No nonreproducible sequ ence alterations were found in frozen tissues. In formalin-fixed material u p to one mutation artifact per 500 bases was recorded. The chance of such a rtificial mutations in formalin-fixed material was inversely correlated wit h the number of cells used in the PCR-the fewer cells, the more artifacts, A total of 28 artificial mutations were recorded, of which 27 were C-T or G -A transitions. Through confirmational sequencing of independent amplificat ion products artifacts can be distinguished from true mutations, However, b ecause this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-f ixed material, including those inserted into mutation databases.