Figure 1

A schematic diagram of the subcloning procedure. A. Sequencing of TP53 cDNA fragment containing exons 4 – 8, that specify which allele with cytosine or guanine in codon 72 (exon 4) is mutated within exons 5 – 8. B. The samples containing an exonic mutation and codon 72 heterozygosity were subjected to bacterial subcloning. A fragment of TP53 gene comprising intron 3 and codon 72 from the selected samples were cloned into a bacterial vector and sequenced. Such an analysis allowed for the sequencing of each allele separately, and therefore, for the detection of haplotypes (i.e. which codon 72 variant co-localized with which PIN3 variant). C. Combination of these results allows to infer which PIN3 allele (A1 or A2) is the mutated one.