Cloning of an overexpression vector for the rare and uncharacterized KRAS mutant R164L

Mutations in the coding DNA of KRAS, which encodes for an active protein in the Ras signaling pathway regulating cell proliferation and growth, may be critical in the development of lung and colorectal cancer in humans. Clinical tests and therapies for cancer specifically target mutations, some of which may cause erroneous prognosis and prescription of cancer therapy posing more danger for patients with unique mutations. Therefore, characterization of non-hotspot mutations is becoming more important, where, KRAS R164L is an existing uncharacterized mutation identified through the Catalogue of Somatic Mutations in Cancer (COSMIC). The project aimed to clone a pTarget overexpression vector for the noncanonical KRAS mutant R164L. Splicing via overlapping extension – PCR (SOE-PCR) was first applied to amplify the KRAS coding sequence and to introduce the guanine to thymine point-mutation in codon 491 of the DNA. The genetic transcript of the KRAS mutant R164L was ligated into the pTarget vector, through the Universal TA cloning technique. A 6kb-long plasmid containing the KRAS R164L mutation was produced, subjected to transformation of E. coli DH5alpha cells via heat-shock method, and screened via Blue-White Screening. The successful transformants were verified through Restriction Enzyme digestion with EcoRI. The DNA sequence of the inserts in the successfully cloned plasmids was further verified through Sanger Sequencing and sequence alignment through NCBI Blast, yielding a 99% match with the coding sequence for Homo sapiens Ras family for KRAS. The DNA segment for KRAS mutation R164L was successfully cloned and may now be utilized in functional characterization assays.