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.
-Prior to inserting the STR sequences into K-ras DNA, the researchers perform MCA analysis using only the double-stranded STR sequences. Which sequence from Table 1 would have the fastest reannealing time in this experiment?

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.
-Prior to inserting the STR sequences into K-ras DNA, the researchers perform MCA analysis using only the double-stranded STR sequences. Which sequence from Table 1 would have the fastest reannealing time in this experiment?

Quizplus · Accepted Answer

11ecba2d_0e71_17d9_a3bf_816fbff27825_MD0008_00 Melted DNA strands will reanneal to form the double helix once temperature decreases, but the time it takes for complementary strands to reanneal depends on the following: DNA length: Longer DNA molecules have more hydrogen bonds and will take more time to both melt and reanneal. pH: The physiological pH range (7.3-7.4) allows maximal hydrogen bonding between nitrogenous bases of DNA. At low pH, hydrogen bond acceptor atoms in the bases become protonated, and these protonated acceptors cannot form hydrogen bonds, causing double helix separation. In contrast, high pH causes deprotonation of hydrogen bond donors in the bases; the loss of protons results in the loss of hydrogen bonds and a destabilized double helix. Salt concentration (ionic strength): The electrostatic repulsion between negatively charged phosphate groups on the sugar-phosphate backbone destabilizes the double helix. However, this repulsion is neutralized and shielded by the binding of positively charged species in solution (eg, Na⁺ and Mg²⁺ cations). High salt concentration of the solution increases double helix stability, but low salt concentration decreases stability. The four short tandem repeats sequences shown in Table 1 were denatured during melting curve analysis and cooled in environments with different pH and NaCl concentrations. Sequence 3 will have the fastest reannealing time as it is the shortest sequence and was allowed to cool in the solution closest to physiological pH with a high salt concentration (>0.15 M). (Choices A and B) Sequences 1 and 2 were studied in solutions with high pH and low salt concentration. Alkaline pH causes a loss of base pairing interactions, and low salt concentration would induce greater repulsion between negatively charged phosphate groups on DNA. In addition, sequence 2 is the longest and would take longer to reanneal. (Choice D) Sequence 4 DNA has a higher number of repeats than sequence 3 and is likely to be destabilized by the extremely acidic pH despite the high salt concentration. Educational objective: The following parameters influence the thermodynamic stability of the DNA duplex: DNA length: Longer DNA molecules take more time to both melt and reanneal. pH: Extreme changes in pH outside the physiological range lead to loss of hydrogen bonding and destabilize the DNA helix. Salt concentration (ionic strength): High salt concentration of the solution increases double helix stability, but decreased salt concentration decreases stability.

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