Question 1

Which of the following molecules polymerize to form a support medium through which nucleic acids move?

Accepted Answer

Polyacrylamide is commonly used in gel electrophoresis as a support medium to separate nucleic acids based on size. When polymerized, polyacrylamide forms a porous matrix that allows small molecules like nucleic acids to move through it, while larger molecules get trapped and separated based on their size. Ethidium bromide is a fluorescent dye used to visualize nucleic acids in the gel, but it does not contribute to the support matrix. Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that can bind to metal ions and is often used as a component of buffer solutions, but it does not contribute to the gel matrix. Sodium dodecyl sulfate (SDS) is a detergent used to denature and linearize proteins for separation in SDS-PAGE, but not for nucleic acids in gel electrophoresis.

Question 2

What concentration of agarose should be used for optimal separation of DNA fragments of 100, 250, and 350 bp in length?

Accepted Answer

A 2.0% agarose gel is suitable for separating small DNA fragments in the range of 100 to 500 bp, providing good resolution for the sizes mentioned.

Question 3

What is an advantage of agarose over polyacrylamide gels?

Accepted Answer

Agarose monomers are not toxic, while acrylamide monomers are toxic and potentially carcinogenic. Therefore, agarose is safer to use in experiments.

Question 4

Which of the following molecules will migrate fastest in capillary electrophoresis?

Accepted Answer

In capillary electrophoresis, smaller molecules migrate faster than larger molecules. Additionally, negatively charged molecules migrate towards the positive electrode, while positively charged molecules migrate towards the negative electrode. Therefore, the molecule that will migrate fastest is small and negatively charged (option D).

Question 5

A very limited amount of nucleic acid, 500-1500 bp in size, is to be analyzed in a short time (same day) with the results available immediately. Which of the following electrophoresis procedures will satisfy those conditions?

Accepted Answer

Capillary electrophoresis is known for its high resolution and speed, making it a good choice for analyzing small fragments of nucleic acid in a short time. It uses a narrow capillary tube and an electric field to separate molecules based on their size, charge, and shape. The results can be obtained immediately after the electrophoresis run. The other three options listed are not suitable for a quick analysis of small DNA fragments.

Question 6

Which of the following molecules is added to an electrophoresis buffer for the purpose of denaturing DNA?

Accepted Answer

Formamide is a strong denaturant that disrupts hydrogen bonding between the bases of double-stranded DNA, thereby causing strand separation. Ethylenediaminetetraacetic acid (EDTA) is not a denaturant; it is added to chelate metal ions that may interfere with DNA migration during electrophoresis. Polyacrylamide is a sieve-like material that separates DNA fragments by size and is commonly used in polyacrylamide gel electrophoresis (PAGE). Tris borate is a buffer that maintains a stable pH and ionic strength during electrophoresis, but it is not a denaturant.

Question 7

Nucleic acids migrate toward which pole in electrophoresis?

Accepted Answer

Nucleic acids, such as DNA and RNA, are negatively charged molecules due to their phosphate backbone. In electrophoresis, they migrate towards the anode, which is the positive pole, because opposite charges attract.

Question 8

How does one prepare a 2% agarose gel?

Accepted Answer

A 2% agarose gel is prepared by mixing 0.02 g of agarose per mL of running buffer and heating the mixture. This will result in a gel with a suitable concentration for electrophoresis of DNA fragments. Options A, C, and D are incorrect because they either use too little or too much agarose per mL of buffer, which will result in a gel with an improper concentration for DNA electrophoresis.

Question 9

Which of the following is not an example of pulse-field gel electrophoresis?

Accepted Answer

Pulse-field gel electrophoresis refers to a specific type of gel electrophoresis technique that uses a series of electrical pulses with varying field strengths and directions to separate large DNA fragments. Polyacrylamide gel electrophoresis, on the other hand, is a different type of gel electrophoresis that is not considered pulse-field gel electrophoresis. A, B, and D are all examples of pulse-field gel electrophoresis techniques.

Question 10

Nucleic acids are injected into the capillary in capillary electrophoresis by which of the following injection methods?

Accepted Answer

Nucleic acids are highly charged molecules and therefore are typically introduced into the capillary by electrokinetic injection, which uses an electric field to drive the sample into the capillary. Hydrostatic injection uses hydraulic pressure to inject the sample, pneumatic injection uses gas pressure, and vacuum injection uses negative pressure, but these methods are not typically used for nucleic acid injection in capillary electrophoresis.

Question 11

To increase the density of a sample relative to the density of the buffer prior to loading the sample into a gel, which of the following is added to the sample?

Accepted Answer

Glycerol is a common additive used to increase the density of a sample relative to the density of the buffer. This allows the sample to sink into the gel rather than float on top. Ethidium bromide is a stain used to visualize DNA, formaldehyde is a crosslinker used in protein assays, and Tris acetate EDTA is a buffer used in electrophoresis. None of these would have a significant impact on the density of the sample.

Question 12

The positive pole in electrophoresis is indicated by which color?

Accepted Answer

Red is commonly used to indicate the positive pole or anode in electrophoresis. This convention is often used in gel electrophoresis protocols and publications.

Question 13

A molecular biologist is to separate and analyze a mixture of nucleic acids that are about 100,000 bp in size. Which of the following methods will yield the best results?

Accepted Answer

Rotating gel electrophoresis is suitable for separating large nucleic acids, such as those around 100,000 bp, due to its ability to handle large DNA fragments more effectively than other methods.

Question 14

The matrix in capillary electrophoresis through which nucleic acids pass is:

Accepted Answer

Capillary electrophoresis commonly uses optimized polymer matrices, such as polyacrylamide or polyethylene glycol, that are designed to provide high resolution and reproducibility in separating nucleic acids based on size and charge. Silica and agarose matrices are commonly used in other nucleic acid separation techniques such as gel electrophoresis. Agarobiose is not a commonly used matrix in nucleic acid separation.

Question 15

Polyacrylamide gel properties can be altered by adjusting T and C values. What are T and C in this context?

Accepted Answer

T refers to the total concentration of acrylamide plus cross-linker, which affects the pore size of the gel, and C refers to the percent cross-linker, which influences the gel's rigidity.

Question 16

To separate a mixture of oligonucleotides with a resolution between bands of 1 bp, which of the following methods should be used?

Accepted Answer

Polyacrylamide gel electrophoresis is capable of resolving DNA fragments with a size difference of just one base pair because of its higher resolving power compared to traditional agarose gel electrophoresis.

Question 17

A molecular technologist has run a vertical gel. While analyzing the gel, the technologist notices that the bands in the outer lanes did not run as far as similar-sized bands in the inner lanes, so the bands look like a smile on the gel. What is the explanation for this?

Accepted Answer

The phenomenon described, where bands in the outer lanes do not run as far as those in the inner lanes, resulting in a "smiling" effect, is typically due to uneven heating across the gel. The inner parts of the gel tend to get warmer than the edges during electrophoresis, leading to faster migration of the molecules in the center compared to those at the edges. This temperature gradient is the reason for the uneven migration and the observed effect.

Question 18

As compared with traditional slab gel electrophoresis, which of the following is not an advantage of capillary electrophoresis?

Accepted Answer

The cost of instrumentation and labels is typically higher for capillary electrophoresis as compared to traditional slab gel electrophoresis due to the need for specialized equipment and fluorescent dyes. All of the other options listed, including the ability to analyze smaller amounts of samples, run multiple samples together, and perform real-time sample detection and analysis, are advantages of capillary electrophoresis over traditional slab gel electrophoresis.

Question 19

What is one reason that polyacrylamide gels have higher resolution than agarose gels for small fragments?

Accepted Answer

Polyacrylamide gels have a smaller pore size than agarose gels. The spaces between the polyacrylamide polymer chains are smaller than those between agarose polymers, which means that polyacrylamide gels can separate smaller fragments with higher resolution.

Question 20

Which of the following are used to make wells in a solidifying gel into which samples are loaded?

Accepted Answer

Combs are used to make wells in a solidifying gel. The teeth of the comb create the wells, and samples are loaded into these wells for analysis. Brushes, picks, and shovels are not typically used in gel electrophoresis.

Quiz 5: Resolution and Detection of Nucleic Acids