A multiplex, allele-specific PCR for rapid and sensitive screening of IDH1 R132 mutations. (A) The sequences of the primers. The 3rd last nucleotide was intentionally mutated to avoid background signals (italic and underlined). The last nucleotide matches only to mutant but not wild-type sequences (italic). Wild-type sequence was shown for reference. (B) Schematic representation of our multiplex, allele-specific PCR strategy. The red vertical bar represents the site of R132 mutation. (C) Multiplex PCR on genomic DNA from bone marrow cell of AML patients. The top band (arrow) indicated an internal control band. The bottom band (arrowhead) represented the mutant signal. Samples w291 and FL94 were positive for IDH1 mutation in both sequencing and multiplex methods. FL91 was negative by sequencing but yielded a mutant band in multiplex PCR. 293T represented a complex genomic DNA from this cell line without IDH1 mutation and served as a negative control. Results from other samples were not shown. (D) Mutant signals were evident in samples 789 (UPN 789, at diagnosis), 1134, 1136 (2 samples from this relapsed patient), and 1531 (a sample from another relapsed patient) by multiplex PCR, but sequencing did not reveal mutation in the samples 1136 and 1531. (E) Mutant bands could be seen in patients' DNA diluted with 293T genomic DNA up to 200-fold (top panel). On the other hand, the mutant signal was barely seen in direct sequencing at 20-fold dilution (bottom panel). In our previous report, only 5 types of IDH1 mutation were seen (no R132P).1 (Top panel) One, 10, and 200 mean dilution fold of mutant genomic DNA with 293T DNA. WT indicates 293T genomic DNA alone.
