Detection of biallelic JAK2 mutations in patients with essential thrombocythemia by allele-specific PCR and bacterial cloning of gDNA or cDNA PCR products. (A) Schematic representation of JAK2 cDNA showing the position of the V617F mutations and the intron 14 and exon 19 single nucleotide polymorphisms (SNPs) used in these studies. (B) Granulocyte genomic DNA (gDNA) from patients 1-4 was amplified using a JAK2 V617F mutation-specific and common reverse primer to generate an amplicon encompassing an informative intron 14 SNP (arrowhead), with sequencing of the amplicon showing a JAK2 mutation on both the C- and T-alleles in patient 1, and a JAK2 mutation on the C allele only in patients 2-4. (C) Amplicons generated from cDNA or gDNA were column-purified, ligated into pGem-T Easy (Promega) or TOPO XL (Invitrogen) vectors and used for transformation of competent Escherichia coli bacteria per the manufacturer's instructions. Individual bacterial colonies were then picked and genotyped by sequencing with M13 primers. Analysis of a cDNA amplicon, encompassing JAK2 exons 14 to 19, by PCR amplification and bacterial cloning suggested the presence of biallelic JAK2 mutations in all 4 patients; however, similar analysis of both a gDNA amplicon encompassing JAK2 exons 14 to 19 and a cDNA amplicon encompassing JAK2 exons 6 and 19 indicated the presence of biallelic JAK2 mutations in patient 1 only. The number of bacterial clones with each genotype is indicated on the individual bar charts. (D) Model to explain the formation of chimeric PCR products in a mixed template reaction due to the extension of a partially annealed, mismatched template, giving rise to the artifactual appearance of biallelic mutations. (E) Analysis by bacterial cloning and genotyping of an exon 14-19 cDNA amplicon generated by either 35 or 25 cycles of initial PCR amplification: analysis of the 35-cycle amplicon suggested biallelic JAK2 mutations in all 10 patients, whereas reducing the initial PCR step to 25 cycles indicated biallelic mutations only in patient 1, concordant with the allele-specific PCR/sequencing assay. These data indicate that apparent biallelic JAK2 mutations are likely to reflect generation of chimeric DNA molecules during the later stages of PCR amplification. The number of individual bacterial clones genotyped is recorded above the graph. All ET patients were diagnosed according to criteria recently published by the British Committee for Standards in Haematology.⁴