GRE Subject Test: Biology : Understanding Cloning and PCR

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

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Example Questions

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Example Question #1 : Understanding Cloning And Pcr

A researcher is performing PCR to amplify a sample of DNA. Unfortunately, he forgot to add the DNA primer prior to starting the experiment. Which of the following results is he most likely to observe?

Possible Answers:

The reaction will work, but amplify a region that was not his target

The reaction will be completely unsuccessful

The reaction will work, but at a significantly slower rate

The reaction will work, but the product will contain many undesired mutations

Correct answer:

The reaction will be completely unsuccessful

Explanation:

The primers are absolutely essential for the functionality of a PCR reaction. The heat-resistant DNA polymerases need the RNA primers to anneal before they can begin replicating DNA. Without the primers, there is no way for the DNA polymerase to function and, therefore, no product will be obtained.

Primers are essential for any DNA replication process and are synthesized by primase during in vivo replication. For PCR, primers targeted to specific genes are artificially created and introduced to the sample. Only when the primers are bound will DNA polymerase be recruited to the replication site.

Example Question #2 : Understanding Cloning And Pcr

A student researcher wants to clone the human hemoglobin gene (HBB) into an expression vector so that he can express HBB in mouse cells and observe the resulting phenotype. 

Which sequence of techniques will allow the student researcher to successfully clone the HBB gene?

Possible Answers:

1. Use a restriction enzyme to digest the expression vector

2. Ligate HBB and the digested vector

3. Amplify HBB via PCR

None of the other answers

1. Use a restriction enzyme to digest HBB

2. Amplify the expression vector via PCR 

3. Ligate the digested HBB and vector

1. Amplify HBB via PCR

2. Use a restriction enzyme to digest the expression vector

3. Ligate HBB and the digested vector

1. Ligate HBB

2. Use a restriction enzyme to digest the expression vector 

3. Amplify HBB and the digested expression vector via PCR

Correct answer:

1. Amplify HBB via PCR

2. Use a restriction enzyme to digest the expression vector

3. Ligate HBB and the digested vector

Explanation:

The most logical procedure for the student is to first use PCR to amplify the HBB gene from human genomic DNA with primers that have restriction sites at the ends (this will faciliate ligation to a restriction enzyme digested vector). Next, the expression vector should be digested using a restriction enzyme, and then the digested vector and the amplified HBB gene can be ligated together. The final product will be two segments of the original vector with the HBB gene spliced between them.

Example Question #3 : Understanding Cloning And Pcr

A student researcher wants to change five consecutive base pairs in the middle of a PCR amplified fragment of DNA. What technique is best suited for this experiment? 

Possible Answers:

Electrophoretic mobility shift assay (ESMA)

None of the other answers

Restriction enzyme digestion

PCR mutagenesis 

DNA ligation 

Correct answer:

PCR mutagenesis 

Explanation:

The most suitable technique is PCR mutagenesis. The student will design primers that anneal imperfectly at the desired site of mutagenesis. These primers will contain the new 5-base pair sequence to be inserted in the new strand. One PCR will amplify a fragment containing the new mutant sequence and all upstream sequences. A second PCR will amplify a fragment containing the mutant sequence and all downstream sequences. Finally, a PCR will amplify one sequence from the two templates, using the original primers used to amplify the full-length fragment. 

Example Question #4 : Understanding Cloning And Pcr

Polymerase chain reaction (PCR) employs a heat-stable polymerase, such as Taq polymerase, to assemble the amplified strand of DNA. Which of the following best describes why heat-stable polymerases are ideal for PCR?

Possible Answers:

Heat-stable polymerases are typically much cheaper than normal polymerases and are therefore more suited to large scale laboratory amplification of DNA. 

Heat-stable polymerase cannot break the double-stranded DNA unless the temperature is very high, thus requiring heat-stable polymerases. 

The interaction between bivalent cations and polymerases is most efficient during high temperature phases, thus requiring heat-stable polymerase. 

PCR requires thermal cycling, and heat-stable polymerases will neither denature nor lose efficacy in DNA synthesis during the high-temperature cycles. 

PCR requires thermal cycling, and heat-stable polymerases can be inactivated when they are not needed during the low temperature phases. 

Correct answer:

PCR requires thermal cycling, and heat-stable polymerases will neither denature nor lose efficacy in DNA synthesis during the high-temperature cycles. 

Explanation:

Polymerases are simply enzymes, and enzymes often denature at high temperatures. If a normal polymerase were used, it would become denatured during every high-temperature phase, which is required for breaking the double-stranded DNA into single-stranded templates. By using heat-stable polymerase, the enzyme will not denature, and there is no need to add new polymerase after every step of the cycle. The DNA strands will continue to be amplified by the heat-stable polymerase originally added. 

Example Question #5 : Understanding Cloning And Pcr

What type of instrument is generally used to complete a PCR amplification?

Possible Answers:

Thermocycler

Fluorometer

Centrifuge

UV Spectrometer

Genetic Analyzer

Correct answer:

Thermocycler

Explanation:

A thermocycler is used to complete a PCR amplification. The thermocycler can be set to cycle through the denaturation, annealing, and extension stages in a determined amount of cycles based on the reaction volume. 

Example Question #5 : Understanding Cloning And Pcr

What are the three steps to PCR thermocycling?

Possible Answers:

Extension, denaturation, annealing

Denaturation, extension, annealing

Denaturation, annealing, extension 

Annealing, denaturation, extension

Annealing, extension, denaturation 

Correct answer:

Denaturation, annealing, extension 

Explanation:

The steps of thermocycling involves denaturation, annealing and extension.

Denaturation is done at a high temperature generally around 94-98°C to break apart the hydrogen bonds of double stranded DNA into single strands. 

Annealing is the second step of thermocycling completed at 50-65°C.  This step must be done at a temperature low enough allowing primers to attach to the single strand, but high enough that hybridization is specific. 

The extension or elongation step of thermocycling is used to allow the DNA polymerase to create the new complementary strand to the template DNA strand. Temperature depends on the DNA polymerase being used but generally ranges from 75-80°C.

Prior to the stages of thermocycling there is a often a hot start step to activate the polymerase if necessary. Following the stages of thermocycling is a final extension stage and final hold stage.

Example Question #6 : Understanding Cloning And Pcr

Majority of the time PCR amplifications include an Initialization step, also known as the Hot Start. What is the purpose of the Hot Start?

Possible Answers:

Denaturation of the DNA strands

Adenylation of the new DNA strands by DNA polymerase

Extension of the template strand

For primers to begin binding to the template strand

Activation of the DNA polymerase

Correct answer:

Activation of the DNA polymerase

Explanation:

The Initialization Step or Hot Start occurs prior to thermocycling in order to activate the DNA polymerase. This is a 1 to ten minute incubation at 95°C. The purpose of using a DNA polymerase that requires a hot start activation is to prevent non-specific amplification products. 

Primers begin binding to the template strand during the annealing step. Extension of the DNA strand occurs later in the thermocycling process. Adenylation of the new strand by DNA polymerase occurs during a final elongation step after the thermocycling rounds. DNA double strands are broken apart in the denaturation step of thermocycling. 

Example Question #8 : Understanding Cloning And Pcr

Which of the following is not a component of a polymerase chain reaction mix?

Possible Answers:

Buffer solution

DNA template strand

Formamide

DNA polymerase

dNTPs

Correct answer:

Formamide

Explanation:

The major eight components of a polymerase chain reaction are: DNA polymerase, buffer solution, primers, magnesium chloride, dNTPs (adenine, guanine, cytosine, thymine), the deoxynucleotide triphosphates, DNA template strand, water, and bovine serum albumin (BSA) (optional).

Example Question #9 : Understanding Cloning And Pcr

After four cycles of thermocycling, how many copies of the targeted region will be in the PCR product?

Possible Answers:

16

8

64

2

32

Correct answer:

16

Explanation:

PCR increases DNA product exponentially. 2 parents strands of DNA are split in the first cycle and amplified into 2 copies of DNA. The second cycles results in amplification of both of the first cycle products. The amount of strands doubles giving you 4 copies after the second cycle. The pattern continues and the amplificed product doubles with each cycle. 

First cycle: 2 copies

Second cycle: 4 copies (22)

Third cycle: 8 copies (23)

Fourth cycle: 16 copies (24)

Fifth cycle: 32 copies (2)

Example Question #7 : Understanding Cloning And Pcr

Which is not a characteristic of a multiplex PCR reaction?

Possible Answers:

All of these

Primers involved in a multiplex PCR reaction must have similar annealing temperatures

Multiplex PCR is comparable to performing several different PCR reactions in one reaction

Multiplex PCR reactions target multiple different regions of DNA template for amplification

Multiplex PCR reactions incorporate a cocktail of different primer sets

Correct answer:

All of these

Explanation:

All of these answers are characteristics of multiplex PCR reactions. A multiplex reaction is meant to simultaneously amplify several different target regions of DNA utilizing several sets of primers. Incorporating several different primer sets is similar to performing multiple PCR reaction all in one tube. The advantage being that less reagents are used and the process is less time consuming.  Because all of the primers are acting in one reaction tube they must be optimized to perform at similar annealing temperatures and under similar conditions. 

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