PCR artifacts may mimic BCR-ABL1 compound mutations in CML patients. (A) Outline of the experimental procedure used. Experiment type 1 controls for recombination during cloning into Escherichia coli; mutant BCR-ABL1 plasmids were subjected to PCR amplification individually, and then equal quantities of amplicons of 2 plasmids were mixed, denatured by heating to 95°C for 5 minutes, and cloned. Experiment type 2 mimics amplification of samples from patients with ≥2 mutant BCR-ABL1 clones; equal quantities of 2 plasmids were mixed and subjected to PCR amplification and cloning. Both types of experiments were replicated using 7 mixtures of 5 different plasmids, each containing 4 to 10 KD mutations. Approximately 3000 copies of each plasmid were used as template for each PCR. Unless otherwise specified, first-round PCR (40 cycles) was performed using the Roche Expand Long Template PCR System and the primers 5′-TGACCAACTCGTGTGTGAAACTC-3′ and 5′- TTCGTCTGAGATACTGGATTCCTG-3′, generating ∼1.5 kb amplicons. After cleanup with ExoSAP-IT (Affymetrix), 1 μL of the amplicons was used as template in a second-round PCR (40 cycles) using the primers 5′-GGGCTCTATGGGTTTCTGAATG-3′ and 5′-ATACTGGATTCCTGGAACATTGTTT-3′, generating ∼1.5 kb amplicons containing the BCR-ABL1 KD. Amplified fragments were cloned into pGEM-T Easy (Promega, Madison, WI) and transformed into E coli strain JM109 to minimize E coli–mediated recombination and repair of heterologous DNA. Individual clones (14-37 per mixture) were subjected to Sanger sequencing to reveal the BCR-ABL1 KD sequence within individual amplicons. Clones without artificial recombination are those where the KD mutations resemble those in either of the plasmids in the mixture, and conversely clones with artificial recombination are those with KD mutations originating from both of the plasmids in the original mixture. (B) The KD mutations potentially generated in clones if artificial recombination did not (i, iii) or did (ii, iv) occur. Circles represent compound mutations in plasmid X, and boxes represent compound mutations in plasmid Y; hexagons represent E255K (mix 1, first patient), and stars represent T315I (mix 1, second patient). (C) Sanger sequencing chromatograms showing KD mutations present in a representative plasmid X (black circles) and plasmid Y (gray boxes) and a clone generated using the experiment type 2 procedure. This clone contained 2 of 3 mutations originating from plasmid X as well as 1 mutation originating from plasmid Y. The artificial recombination event occurred within the region marked by the hashed bar. (D) Artificial BCR-ABL1 compound mutations are generated by PCR amplification of mock samples created by mixing equal quantities of cDNA from 8 different CML patients (analogous to experiment type 2; 3 mixtures of 2-3 patient cDNA samples each). Where human tissue was involved, research was conducted with institutional ethics review board approval and in conformance with the Declaration of Helsinki. Mutations present in the original patient samples are shown in bold. Mutations present in >1 clone, but not detected by Sanger sequencing or mass spectrometry in the individual patient samples (“additional mutations”), are shown in regular text; with the exception of exon 7 deletion, these mutations have not been reported in CML patients and likely represent artifacts generated by inaccurate nucleotide incorporation by the DNA polymerase. The frequency of each clone is shown in proportion to the number of clones sequenced per mixture. A total of 13 different clones were detected for mix 1, 5 different clones for mix 2, and 7 different clones for mix 3.