Last updated: 24 October 2024
An article by Prior et al., 2012 has useful information (which otherwise can be difficult to find) about K-Ras and how various mutations alter the function of the protein - e.g., 'activating' mutations in Glycine 12 (G12), G13, Q61, as discussed in the main article. Prior et al., 2012 abstract
Reference (in the 376 literature folder): Prior IA, Lewis PD, Mattos C (2012) A Comprehensive Survey of Ras Mutations in Cancer. Cancer Research 72: 2457-2467.
Terminology note: mutation 'G12R' means a base change in the DNA alters codon 12 from the wildtype encoded amino acid 12 Glycine (G) to Arginine (R). In this case, the mutation converts the wildtype proto-oncogene KRAS into an oncogene.
Key information from the Prior article: 99% of all cancer-promoting mutations in ras genes (encoding one of the 3 different RAS proteins: KRAS, HRAS, NRAS) occur in codons 12, 13 & 61, encoding amino acids G12, G13, and Q61 (G = glycine, Q = glutamine). This region of the RAS protein is identical in KRAS, HRAS, and NRAS, and these amino acids are involved in binding GDP / GTP (schematic of RAS structure - part of Prior Figure 1). [As a matter of interest: Prior Figure 2 - RAS mutation bias in various cancer types.]
Recall that RAS is a 'G protein' - a Guanosine nucleotide-binding protein, and that the activated form is RAS-GTP. (inactive form RAS-GDP). Changes in amino acids G12, G13, and Q61 inhibit the hydrolysis of GTP to GDP (RAS inactivation), locking RAS into the 'activated' form (RAS-GTP).
Read the Materials & Methods sections relevant to the results you are presenting. You should understand this and be able to explain it. Nevertheless, your presentation should NOT be focussed primarily on techniques, but rather the results and discussion. Explain the methods only as much as is necessary to make the results understandable.
Note that the full methods for Misale et al., 2012 are not found in the PDF - you must go to the online version to find the full methods (to access from off campus you will have to login to your MySandiego account, etc. Get USD library help if you don't know how to do this, or access while on campus).
Or, you can access a copy of the full methods, although none of the links will work; this is simply copied as HTML from the Nature website.
Note that you may want or need to show supplemental figures & tables in your presentation. This file is also found in the papers folder, entitled 'Misale2012_Supplemental_info.pdf'
FISH analysis - Fluorescent In Situ Hybridization, used to detect genes on chromosomal spreads of tissue such as tumor tissue to evaluate chromosomal abnormalities such as translocations & other rearrangements, duplications, and individual gene loss or amplification.
This paper uses a number of next-generation high throughput DNA sequencing techniques to sequence much or all of the expressed genome (the 'exome') and to detect low-frequency differences in DNA sequences (that is, different sequences found in a fraction of cells in a tissue). Exome sequencing is used here, e.g., to show amplification of genes (extra copies) such as EGFR in 'DiFi' cells, and KRAS in 'DiFi-R' cells (Fig. 1 b).
BEAMing - individual DNA molecules are associated with magnetic beads in an aqueous droplet in oil (an 'emulsion'), and amplified to have thousands of copies of the identical sequence from the original molecule. These sequences are then determined from these beads - this allows rare sequences to be more reliably detected among thousands to millions of sequences. BEAM - bead, emulsion, amplification, magnetic.
454 pyrosequencing - another high throughput next-generation sequencing method. (Note: this particular method is obsolete; the company Roche stopped making the machines and supporting the technology in the mid-2010's.)
