Passage
The K-ras gene codes for a small guanine nucleotide-binding protein, which acts as a molecular activator in numerous signaling pathways that promote DNA replication along with cell growth and survival. K-ras mutations at codons 12 and 13 function as biological markers for diagnosis of tumors. The DNA of SW-480 cells, a line originating from a colon adenocarcinoma, is the standard positive control used to identify codon 12 mutations in K-ras testing.
Figure 1 Structural comparison of K-ras codon 12 in wild-type cells and SW-480 cellsPCR sequence analysis has been the preferred method for studying genetic mutations such as those in K-ras. However, challenges in analyzing PCR amplicons have prompted the development of DNA melting curve analysis (MCA) . In the MCA technique, DNA is extracted from clinical samples and amplified by PCR. The amplified DNA is then melted (30-70°C) and analyzed by assessing the fluorescence (at 640 nm) of LC Green Plus, an intercalating dye that fluoresces when bound to double-stranded DNA (Figure 2) .
Figure 2 Melting curves of wild-type (WT) and SW-480 K-ras DNA.To study K-ras activity in colorectal cancer cells, short tandem repeats (STRs) were introduced into the K-ras gene. STRs are short stretches of DNA with repeating nucleotides, and four different STR sequences were inserted into the K-ras gene and analyzed via MCA under different experimental conditions (Table 1) .Table 1 Sequences Studied in K-ras DNA MCA Analysis.
Liu YP, Wu HY, Yang X, et al. Diagnostic accuracy of high resolution melting analysis for detection of KRAS mutations: a systematic review and meta-analysis. Sci Rep. 2014;4:7521.
-Assuming that additional mutations similar to the one shown in Figure 1 occur throughout the mutant K-ras gene, what would be the best explanation for the difference in denaturation between wild-type (WT) and mutant K-ras DNA?

Question

Passage
The K-ras gene codes for a small guanine nucleotide-binding protein, which acts as a molecular activator in numerous signaling pathways that promote DNA replication along with cell growth and survival. K-ras mutations at codons 12 and 13 function as biological markers for diagnosis of tumors. The DNA of SW-480 cells, a line originating from a colon adenocarcinoma, is the standard positive control used to identify codon 12 mutations in K-ras testing.

Figure 1 Structural comparison of K-ras codon 12 in wild-type cells and SW-480 cellsPCR sequence analysis has been the preferred method for studying genetic mutations such as those in K-ras. However, challenges in analyzing PCR amplicons have prompted the development of DNA melting curve analysis (MCA) . In the MCA technique, DNA is extracted from clinical samples and amplified by PCR. The amplified DNA is then melted (30-70°C) and analyzed by assessing the fluorescence (at 640 nm) of LC Green Plus, an intercalating dye that fluoresces when bound to double-stranded DNA (Figure 2) .

Figure 2 Melting curves of wild-type (WT) and SW-480 K-ras DNA.To study K-ras activity in colorectal cancer cells, short tandem repeats (STRs) were introduced into the K-ras gene. STRs are short stretches of DNA with repeating nucleotides, and four different STR sequences were inserted into the K-ras gene and analyzed via MCA under different experimental conditions (Table 1) .Table 1 Sequences Studied in K-ras DNA MCA Analysis.

Liu YP, Wu HY, Yang X, et al. Diagnostic accuracy of high resolution melting analysis for detection of KRAS mutations: a systematic review and meta-analysis. Sci Rep. 2014;4:7521.
-Assuming that additional mutations similar to the one shown in Figure 1 occur throughout the mutant K-ras gene, what would be the best explanation for the difference in denaturation between wild-type (WT) and mutant K-ras DNA?

Quizplus · Accepted Answer

11ecba2d_0e70_2d78_a3bf_ebe5591c340f_MD0008_00 The GC content is the percentage of guanine and cytosine bases in DNA, and the higher the GC content, the greater the stability and melting temperature (T_m) of double-stranded DNA. DNA double helix stability is primarily maintained by stacking interactions: the interactions between neighboring G≡C base pairs are stronger than those between adjacent A=T base pairs. It also takes more energy (heat) to break the three hydrogen bonds in G≡C base pairs than the two hydrogen bonds in A=T base pairs. As a result, the T_m for GC-rich DNA molecules is higher as additional energy is required to separate the more stable helix. Figure 1 shows that SW-480 cells have a G→T base substitution at codon 12 of the K-ras gene, depicting a loss in GC content. Assuming there are additional similar mutations in the mutant K-ras gene, the mutant DNA will have lower GC content than the WT DNA and therefore be less stable and denature faster (ie, exhibit lower T_m). (Choice A) Mutant K-ras DNA with various mutations similar to the one shown in Figure 1 would exhibit a loss of GC content and decreased stability and T_m. (Choices C and D) WT DNA and mutant K-ras DNA are the same length because the mutation in K-ras from SW-480 cells is a base substitution and not an insertion or deletion. Longer DNA molecules have increased T_m because they have more base pairs that stabilize the double helix and require more energy to be broken. Educational objective: G≡C base pairs form more hydrogen bonds and have stronger stacking interactions than A=T base pairs. The higher the GC content, the greater the melting temperature (T_m) and stability of double-stranded DNA.

Passage The K-Ras Gene Codes for a Small Guanine Nucleotide-Binding Protein