GRE Subject Test: Biology : Understanding Separation Techniques

Study concepts, example questions & explanations for GRE Subject Test: Biology

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Example Questions

Example Question #2 : Organic Analyses And Lab Techniques

When would it be appropriate to use extraction in order to separate compounds in a solution?

Possible Answers:

When the compounds have similar molecular weights, but differing polarities.

When the compounds have differing molecular weights, but similar solubilities.

When the compounds have similar polarities, but differing solubilities.

When the compounds have differing conjugated double bond lengths, but similar molecular weights.

Correct answer:

When the compounds have similar polarities, but differing solubilities.

Explanation:

Extraction is a useful separation technique when there is a mixture of compounds in a solution that have similar polarities, but different solubilities. The three-step process of extraction can take advantage of different solubilities by introducing the mixture to different acidic and basic conditions.

Example Question #1 : Understanding Separation Techniques

In polymerase chain reaction (PCR), the reaction mixture is heated to approximately 98°C during the first cycling step in the procedure. Which of the following describes the purpose of this step?

Possible Answers:

Degradation of non-target sequences within the template

Activation of Taq polymerase

Bringing the reaction to the optimal temperature for annealing of the primers to the template

Melting of the DNA template, producing single-stranded DNA to which primers can bind

Solubilizing the dNTPs

Correct answer:

Melting of the DNA template, producing single-stranded DNA to which primers can bind

Explanation:

Heating of the reaction to roughly 98°C is required to separate the template DNA strands from one another, thereby producing single strands that can pair to their complementary primer. At this temperature, the hydrogen bonds between base pairs break and the strands separate.

Taq polymerase doesn't require this temperature to be active, non-target sequences are not degraded during PCR, the dNTPs are already soluble, and the optimal annealing temperature for primers is actually much lower than 98°C. 

Example Question #2 : Understanding Separation Techniques

Which technique is best suited to determine if a protein is active and able to bind DNA? 

Possible Answers:

Northern blot

Western blot

Electrophoretic mobility shift assay (EMSA) 

Southern blot

Bradford assay

Correct answer:

Electrophoretic mobility shift assay (EMSA) 

Explanation:

In an EMSA, a radiolabeled sequence of DNA that the protein of interest normally binds is incubated with the protein. This mixutre is then run on a non-denaturing gel. If the protein binds the radiolabeled DNA sequence, radioacitivity will be detected towards the top of the gel; however, if it does not bind, the radiolabeled DNA probe alone will run more quickly towards the bottom of the gel. 

Example Question #3 : Understanding Separation Techniques

What is the purpose of a electrophoretic mobility supershift assay? 

Possible Answers:

To separate proteins by size

To determine if two proteins interact with one another

To separate proteins by charge

 

To determine if a certain protein binds a DNA sequence

To separate DNA fragments by size

Correct answer:

To determine if a certain protein binds a DNA sequence

Explanation:

The purpose of an electrophoretic mobility supershift assay (EMSA) is to determine if a certain protein binds a DNA sequence. EMSAs determine if a protein is "active", meaning that it is capable of binding its target DNA sequence. In an EMSA, a radiolabelled DNA fragment is incubated with cellular protein lysates, then run on a non-denaturing gel. Any protein-bound DNA fragments will migrate slower than unbound DNA fragments. When performing a supershift, one wants to determine if a specific protein binds a radiolabelled DNA probe by use of an antibody. If the antibody binds the protein-DNA complex, this will migrate even slower than the protein-DNA complex alone. 

Example Question #4 : Understanding Separation Techniques

Capillary electrophoresis instrumentation involves which of the following components?

Possible Answers:

Positive and negative electrodes 

All of these

Polymer

Detector

Buffer system

Correct answer:

All of these

Explanation:

A buffer system is required in a capillary electrophoresis system in order to supply the ions necessary to carry the electric current. Ions become depleted quickly and the buffer system will need to be replenished regularly.

The positive and negative electrodes, in combination with a high voltage power supply create the current. The source of the sample and destination of the sample will have opposite charged electrodes. Migration of the analytes through the polymer filled capillary will depend on the applied electric field and their size.

The detector of a capillary electrophoresis system will vary based on the instrumentation type. It is used to detect the size of the molecules and the speed at which they move through the matrix. Sizing methods and databases are then used for analysis.

Polymer serves as the separation matrix of the system. The viscosity of the polymer in combination with the electroosmotic flow of they system will separate molecules based on their size. 

Example Question #5 : Understanding Separation Techniques

Compared to slab gel electrophoresis, which is an advantage of capillary electrophoresis?

Possible Answers:

All of these

Less sample consumption

Reproducibility of matrix viscosity

Data collection can be done in real-time

Automatable sample loading

Correct answer:

All of these

Explanation:

Capillary electrophoresis allows for better reproducibility of liquid polymer throughout a capillary compared to slab gels. Much of the time gels are manually poured and uneven gel thickness can occur. 

Real time data viewing is possible on a capillary electrophoresis instrument's system computer. 

Higher sample consumption is common with slab gels. More sample is required to be loaded in each lane. If retesting is necessary, the sample must be prepared and loaded into a new gel. Very small quantities of sample are consumed in the injection step into the capillary. Samples can be easily retested through reinjection from the original sample vial.

Capillary electrophoresis has the possibility of being a completely automated process, including automated sample loading. This saves analyst time during sample prep, injection, and separation. Gels require manual sample loading and some instruments require gel handling for scanning or photography after the electrophoresis process. 

Example Question #6 : Understanding Separation Techniques

Which of the following is a method for sample introduction into a capillary electrophoresis system?

Possible Answers:

Siphoning

Pressure injection

All of these

Electrokinetic injection

Hydrodynamic injection

Correct answer:

All of these

Explanation:

All of these methods require the immersion of the capillary end into the sample for introduction into the capillary electrophoresis system.

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