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.
-What are the physical states of wild-type (WT) and SW-480 K-ras DNA at 55°C?

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.
-What are the physical states of wild-type (WT) and SW-480 K-ras DNA at 55°C?

Quizplus · Accepted Answer

11ecba2d_0e6f_1c07_a3bf_c5aecf477c3f_MD0008_00 Figure 2 shows the DNA melting curve for wild-type (WT) K-ras DNA and mutant K-ras DNA produced by SW-480 cells. To generate a melting curve, identical copies of each type of double-stranded DNA are gradually exposed to increasing temperatures, which causes the separation (denaturation) of the double helix into two strands of DNA. The melting temperature (T_m), depicted as the midpoint of the melting curve, is achieved when 50% of double-stranded DNA has become single-stranded (ie, when half of the helical structure is lost). LC Green Plus is a compound that fluoresces strongly when bound to double-stranded DNA but has minimal fluorescence with single-stranded DNA. The amount of double-stranded DNA in a sample can be detected by measuring the fluorescence of LC Green Plus. Consequently, as temperature increases in DNA melting curves, the fluorescence decreases as double-stranded DNA becomes single-stranded. In Figure 2, both WT K-ras and SW-480 K-ras DNA have a maximal fluorescence of 0.04 at 20°C, which signals that all DNA molecules are double-stranded and bound by LC Green Plus. The fluorescence of WT K-ras DNA decreases to 0.02 at 55°C, which is the T_m of WT K-ras DNA and signifies that 50% of the WT DNA has melted and become single-stranded while the other 50% is double-stranded. However, mutant K-ras DNA from SW-480 cells reacts differently at 55°C; the fluorescence decreases to a value near zero, indicating that 100% of mutant K-ras DNA is single-stranded. (Choices A, C, and D)At at 55°C, Figure 2 shows that 100% of SW-480 K-ras DNA is single-stranded (not double-stranded) but that 50% (not 100%) of WT DNA is single-stranded. Educational objective: To produce a melting curve, double-stranded DNA is gradually exposed to increasing temperatures, which cause the separation of the double helix into two strands of DNA. The melting temperature (T_m) is achieved when 50% of double-stranded DNA has become single-stranded; T_m is usually depicted as the midpoint of the melting curve.

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