GRE Subject Test: Biochemistry, Cell, and Molecular Biology : Help with Identification Techniques

Study concepts, example questions & explanations for GRE Subject Test: Biochemistry, Cell, and Molecular Biology

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Example Questions

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Example Question #1 : Help With Identification Techniques

Of the following choices, which technique would be the most useful for identifying the binding site of a recently discovered transcription factor?

Possible Answers:

Western blotting

DNA footprinting

Northern blotting

PCR

Correct answer:

DNA footprinting

Explanation:

One function of DNA footprinting involves looking for binding sites by using nucleases to cleave DNA. Wherever the transcription factor is bound should be blocked and protected from cleavage. A sample of DNA can be run in a gel without transcription factors bound and compared to a gel run with DNA that has been exposed to the transcription factor. The difference in DNA sequence can be analyzed based on cleavage differences to identify the transcription factor binding region.

Northern blotting helps identify gene expression by looking at RNA. Western blotting helps identify specific proteins in a sample. PCR helps amplify a sample of DNA.

Example Question #1 : Help With Identification Techniques

In a western blot, to what does the secondary antibody bind?

Possible Answers:

Primary antibody

Nitrocellulose membrane

Protein of interest

Ponceau S dye 

Correct answer:

Primary antibody

Explanation:

The secondary antibody is designed to interact specifically with the primary antibody. This is a crucial step because the secondary antibody will contain a detectable marker (usually a fluorescent marker).

The primary antibody interacts with the protein of interest. The membrane is blocked with milk or some other substance prior to addition of the antibodies to prevent nonspecific binding. Ponceau S dye is used to quickly and reversibly detect quantities of proteins on nitrocellulose membranes.

Example Question #3 : Help With Identification Techniques

A student researcher in a lab has just discovered a new species of bacteria. Since very little is known about this species, he decides to execute an experiment to test for peptidoglycan in the outer plasma membrane. He determines that the bacteria does not contain an outer layer of peptidoglycan.

What experiment did the student perform? Is this bacteria virulent?

Possible Answers:

There is not enough information to deduce the experiment the student performed or determine the virulence of the bacteria

The student performed a gram stain and the bacteria is not virulent 

The student performed a Western blot and the bacteria is not virulent

The student performed a Western blot and the bacteria is virulent

The student performed a gram stain and the bacteria is virulent

Correct answer:

The student performed a gram stain and the bacteria is virulent

Explanation:

The correct answer is the student performed a gram stain and the bacteria is virulent. A gram stain specifically stains peptidoglycan, which is in the outer membrane layer of some bacteria, purple. The presence of peptidoglycan marks a bacteria as gram-positive. If peptidoglycan is not present, the bacteria will not stain purple, and the bacteria is said to be gram-negative. Gram-negative bacteria contain an outer layer of lipopolysaccharides that confer antibiotic resistance and virulence. Since we know that the bacteria in question doe not have an outer peptidoglycan layer, we know that it is virulent.

Example Question #2 : Help With Identification Techniques

Northern, Southern, and Western blots, as originally invented by E. M. Southern, are techniques used in different areas of molecular biology. What can you learn from a Northern blot?

Possible Answers:

The length of DNA transcripts, the number of transcripts isolated, and the expression level

The level of RNase activity

The size of a protein

The length of RNA transcripts, the number of transcripts isolated, and the expression level

The length of DNA or RNA transcripts and the number of transcripts isolated

Correct answer:

The length of RNA transcripts, the number of transcripts isolated, and the expression level

Explanation:

Northern blots are used to study RNA. When run on the gel used in a Northern, you can observe the length of the RNA (by how far it moves on the gel), the number of transcripts (the number of dark bands), and the expression level (how dark the bands are). Southern blots are used to study DNA, and Western blots are used to study proteins.

Example Question #3 : Help With Identification Techniques

A researcher barcodes a library of 3000 plasmids expressing unique growth enzymes and transforms them into bacteria. These 3000 transformed bacteria are then all grown in one large culture of minimal media. Why does the researcher barcode these plasmids? 

Possible Answers:

To determine which enzymes are expressed in the transformed bacteria

To directly drive the transformed bacteria to become quiescent

To directly drive the transformed bacteria to proliferate profusely 

To disrupt the expression of a specific gene in the transformed bacteria

To determine which plasmids promote proliferative bacteria growth

Correct answer:

To determine which plasmids promote proliferative bacteria growth

Explanation:

The correct answer is to determine which plasmids promote proliferative bacteria growth. In this experiment, the researcher wants to identify enzymes that, when over expressed in bacteria, allow the bacteria to grow in unfavorable (minimal) conditions. Since this researcher grows all 3000 transformed bacteria, each expressing a different enzyme, in one minimal media culture, he needs to be able to identify which enzymes promote growth. A barcode is an engineered unique DNA sequence that can be inserted on the plasmid expressing the enzyme. The researcher barcodes each unique plasmid. If a certain enzyme promotes bacteria growth, there will be bacterial replication and replication of the plasmid. Sequencing of the entire bacterial culture will determine which barcodes are overrepresented. Each barcode is then associated with the unique enzymes that allow for proliferative growth in minimal media. 

Example Question #4 : Help With Identification Techniques

A researcher wants to determine if two proteins form a complex in cells. What technique is best suited for this experiment?

Possible Answers:

Co-immunoprecipitation 

TUNEL assay

Electrophoretic mobility shift assay

Protein Binding Microarray

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET)

Correct answer:

Co-immunoprecipitation 

Explanation:

The correct answer is co-immunoprecipitation. During this experiment, protein-protein complexes are cross linked then pulled down by an antibody specific to one of the proteins of interest. Then, these protein elutes are run on a western blot and probed for with a second antibody specific for the other protein believed to be in complex. An electrophoretic mobility shift assay measures the DNA-binding ability of a protein. A TUNEL assay is a measure for apoptosis by quantifying fragmented DNA. Protein binding microarrays determine the preferred DNA binding sequences for a given protein. ChIA-PET measures distal chromatin interactions within the genome. 

Example Question #5 : Help With Identification Techniques

When performing whole-mount immunofluorescence of a specimen, background staining is often a concern with custom generated antibodies. Which of the following is not a way in which to increase specificity of the custom antibody? 

Possible Answers:

Affinity purify the custom antisera

Determine optimal antibody dilution for the application by performing serial dilutions 

Determine optimal blocking buffer components and concentrations 

None of the other answers 

Generate the custom antibody against purified protein rather than a predictive peptide

Correct answer:

None of the other answers 

Explanation:

The correct answer is none of the other answers. To increase specificity of the generated antibody, it is beneficial to expose the animal (in which the antibody will be made) to the entire purified protein, rather than a short synthesized predictive peptide. Moreover, affinity purifying the antibody by exposing the bled antisera to the purified protein and separated the bound antibody-protein fraction from antibody and protein alone will increase specificity. Following antibody generation, determining the optimal dilution of the antibody as well as appropriate blocking conditions to compete out non-specific binding with the specimen are necessary. 

Example Question #6 : Help With Identification Techniques

When is indirect immunofluorescence of sectioned specimens beneficial over whole-mounted specimens? 

Possible Answers:

Sectioning is preferred if the whole-mount is too large in the X plane to resolve by confocal microscopy

Whole-mount staining is only for cryo-preserved specimens

Sectioning is preferred if the whole-mount is too large in the Y plane to resolve by confocal microscopy

Sectioning is preferred if the whole-mount is too large in the Z plane to resolve by confocal microscopy

Sectioning if preferred when using RNA probes, whereas whole-mount is solely for antibody staining 

Correct answer:

Sectioning is preferred if the whole-mount is too large in the Z plane to resolve by confocal microscopy

Explanation:

The correct answer is sectioning is preferred if the whole-mount is too large in the Z plane to resolve by confocal microscopy. Sectioning takes a small slice of an otherwise large specimen, and then subsequent antibody staining can be performed. Typically, this is preferable when one desires to stain certain cell types/layers or the entire specimen is too thick (large Z plane) to mount on a coverslip and resolve by microscopy. 

Example Question #7 : Help With Identification Techniques

A student researcher runs an agarose gel electrophoresis of a restriction enzyme digested plasmid to generate two fragments of 1200 bp and 3500 bp. The student then stains the gel with ethidium bromide. Which of the following is true about the bands that are seen on the gel?  

Possible Answers:

The 1200 bp fragment will be closer to the loading wells than the 3500 bp fragment when the gel is imaged

A simple gel electrophoresis is not sufficient to separate a 1200 bp fragment from a 3500 bp fragment 

The 1200 bp fragment will image more brightly than the 3500 bp fragment 

The 1200 bp fragment will not image as brightly as the 3500 bp fragment

None of the other answers 

Correct answer:

The 1200 bp fragment will not image as brightly as the 3500 bp fragment

Explanation:

The correct answer is the 1200 bp fragment will not image as brightly as the 3500 bp fragment. Ethidium bromide intercalates into DNA proportionally to the size of the fragment. Since we expect the same number of molecules of 1200 bp and 3500 bp fragments, the 1200 bp fragment should be roughly 3 times less bright than the 3500 bp fragment based on the number of ethidium bromide molecules incorporated into the strands. Additionally, smaller fragments migrate more quickly than larger fragments, and as such, are farther from the loading wells. 

Example Question #8 : Help With Identification Techniques

When screening a microenvironment for its bacterial species composition, which genes are most commonly used during molecular typing? 

Possible Answers:

Transcription factor genes 

Transfer RNA genes 

Ribosomal RNA genes 

Mitochondrial genes

DNA polymerase genes

Correct answer:

Ribosomal RNA genes 

Explanation:

The correct answer is ribosomal RNA genes. These genes have highly conserved regions that scientists can use to design primers to amplify intervening regions. These intervening regions, however, are highly divergent and are extremely useful sequences to distinguish between species. 

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