Figure 1
From: Activation of K-RAS by co-mutation of codons 19 and 20 is transforming
Dideoxysequence traces of the L19F and T20A double mutation in K-RAS. (A) Identification of double peaks at the third base of codon 19 and first base of codon 20 in a colorectal cancer. This sequence trace did not determine whether both base changes were on the same or two independent alleles. Harvesting of tumour genomic DNA and sequencing methodology has been described in detail elsewhere [16]. (B) Sequence trace (upper panel), demonstrating the normal leucine and threonine codons at positions 19 and 20 of the K-RAS sequence. Sequence trace from a bacterial clone (lower panel) showing the appearance of the double mutation L19F/T20A in the same allele. For cloning of tumour derived PCR amplicons, products were ligated into TOPOBlunt plasmids using ZeroBlunt TOPO cloning kits (Invitrogen, Paisley, UK) according to the manufacturer's instructions. Bacterial DNA was then isolated using Miniprep kits (Qiagen, Crawley, UK) according to the manufacturer's instructions and sequenced using M13 universal primers on a ABI3730xl Platform sequencer (Applied Biosystems, Warrington, UK).