Section 4

Question 1

How does the PCR procedure work, and what are the key components of a basic PCR reaction mixture?

Answer 1

PCR relies on DNA polymerases, enzymes that use pre-existing DNA templates and free deoxyribonucleotides to synthesize DNA strands. It does not create DNA from scratch but extends primers on existing strands. PCR uses a thermostable DNA polymerase, such as Taq polymerase, which can withstand heating. In a typical PCR reaction, two synthetic primers complementary to target DNA sequences define the amplified segment’s ends. These primers are extended by DNA polymerase. The basic PCR reaction mixture includes the DNA sample, synthetic primers, deoxynucleoside triphosphates (dNTPs), and DNA polymerase.

Question 2

What is the primary function of DNA polymerases in the PCR procedure?

Answer 2

DNA polymerases in PCR are enzymes that synthesize DNA strands using pre-existing DNA templates and free deoxyribonucleotides. They extend primers on existing DNA strands.

Question 3

How does a thermostable DNA polymerase, like Taq polymerase, contribute to the PCR process?

Answer 3

A thermostable DNA polymerase, such as Taq polymerase, can withstand the heating steps of the PCR procedure without denaturing.

Question 4

What is the role of synthetic primers in a PCR reaction, and how are they prepared?

Answer 4

Synthetic primers, complementary to target DNA sequences, define the ends of the amplified segment and can be extended by DNA polymerase. They are prepared synthetically.

Question 5

What are the key components of a basic PCR reaction mixture?

Answer 5

A basic PCR reaction mixture typically includes the DNA sample containing the segment to be amplified, a pair of synthetic oligonucleotide primers, deoxynucleoside triphosphates (dNTPs), and DNA polymerase.

Question 6

How many cycles are typically repeated in the PCR procedure, and what is the outcome of these cycles?

Answer 6

The PCR procedure typically involves repeating the cycle of heating, cooling, and extension 25 to 30 times over a few hours. Each cycle doubles the amount of the amplified DNA segment, resulting in exponential growth. After 20 cycles, the DNA segment can be amplified up to a million times under ideal conditions.

Question 7

What is the purpose of the annealing step in PCR?

Answer 7

The annealing step cools the mixture so that synthetic primers can bind to the DNA template. The high primer concentration increases the chances of primer binding to the denatured DNA strands.

Question 8

What is the main function of the elongation step in PCR?

Answer 8

The elongation step slightly increases the temperature to allow DNA polymerase to synthesize a complementary DNA strand. It extends the strands along the targeted segment.

Question 9

Describe the amplification step in PCR.

Answer 9

In the amplification step, the heating and cooling process is repeated, denaturing the DNA, allowing primers to anneal, and elongating the DNA strands. This step amplifies the target DNA segment.

Question 10

What are the general steps in the PCR procedure?

Answer 10

1. Denaturation: Heat to separate DNA strands.
2. Annealing: Cool to allow primers to bind to DNA.
3. Elongation: Slightly increase temperature for DNA polymerase to synthesize a complementary strand.
4. Amplification: Repeat denaturation, annealing, and elongation to amplify the target DNA segment. This cycle is repeated 25 to 30 times. Each cycle doubles the amount of amplified DNA. After 20 cycles, the DNA segment can be amplified up to 2^20 (a million times) under ideal conditions.

Question 11

You wish to PCR amplify a segment of DNA that is 1900bp in length. What is the minimum extension time required to ensure the successful amplification of this segment?
You can respond once
30 seconds
45 seconds
1 minute
2 minutes
10 minutes

Answer 11

2 minutes

because we are going at a synthesis rate of 1000 base pairs per minute

Question 12

What is the synthesis rate of the PCR reaction?

Answer 12

approx. 1000 bp/min

Question 13

What are the key parameters for designing a good PCR primer set?

Answer 13

18-25 nucleotides in length.

40-60% GC content.

Annealing temperature (Ta) in the range of 50-60°C.

One or two GC residues at the 3’ end of the DNA strands.

Minimal secondary structure and base repeating.

Complementary to the sequence chosen for amplification.

Ta, the ideal annealing temperature, is generally 5°C lower than the melting temperature of the primer.

Question 14

What is the formula for calculating the melting temperature (Tm) of double-stranded DNA fragments, and how does it differ for shorter oligonucleotides?

Answer 14

The formula for calculating Tm for double-stranded DNA fragments is Tm = 0.41 (%G+C) + 69.3°C. For shorter oligonucleotides between 14-20 base pairs in length, you can use the Wallace rule: Tm = 2°C (A+T) + 4°C (G+C). To estimate the annealing temperature (Ta), subtract 5°C from the Tm: Ta = Tm - 5°C.

Question 15

What is an ideal primer sequence used for?

Answer 15

It lands on either side of the exon to amplify the segment in between

Question 16

Every additional base that is added __________ the melting temperature because you are adding extra ____________

Answer 16

Every additional base that is added INCREASES the melting temperature because you are adding extra HYDROGEN BONDS

Question 17

What is the purpose of gel electrophoresis in molecular biology?

Answer 17

Gel electrophoresis is used to separate large charged molecules like nucleic acids or proteins based on size by applying an electric field to a gel matrix.

Question 18

What type of material makes up the gel matrix used in electrophoresis, and why is it chosen?

Answer 18

The gel matrix is composed of agarose, a kelp-derived material that does not disrupt nucleic acid base pairing.

Question 19

How does gel electrophoresis separate molecules in a sample?

Answer 19

When a voltage is applied to the gel, negatively charged nucleic acids like DNA and RNA migrate towards the positive end. Larger molecules move more slowly, while smaller ones move faster, resulting in separation by size.

Question 20

In vitro DNA amplification by PCR differs from in vivo DNA replication in that:
- PCR uses RNA primers
- PCR uses heat to separate the DNA double helix
PCR uses ddNTPs
- PCR uses Mg2+ ions to coordinate reactants in the catalytic core of the polymerase
- PCR uses a heat-sensitive yeast DNA polymerase

Answer 20

PCR uses heat to separate the DNA double helix.

PCR uses heat to separate the DNA double helix instead of relying on helicase. It also uses DNA primers and dNTPs for DNA replication. Unlike the helicase used in living cells, PCR is not sensitive to heat, and this heat resistance is essential for the reaction to work properly.

Question 21

How can you optimize PCR conditions for more selective amplification of your desired product?

Answer 21

1. Increase annealing temperature:
   By raising the annealing temperature, you make it more specific for the primer to bind to the target DNA. This reduces nonspecific binding and increases the selectivity of the amplification.
2. Reduce salt concentration
   - Increasing [MgCl2] = less stringent conditions
   - Decreasing [MgCl2] = sub-optimal polymerase activity
   Salt concentration can influence the stability of DNA strands. Lowering the salt concentration, specifically MgCl2, can create less stringent conditions. This can help make the PCR reaction more selective, as it may decrease nonspecific interactions, but be cautious not to lower it too much, as it can also reduce polymerase activity.

Question 22

In PCR, MgCl2, is added to the reaction mixture to facilitate the DNA amplification process. Why?

Answer 22

Coordinating dNTPs: Magnesium ions (Mg2+) play a crucial role in the catalytic core of DNA polymerases. These enzymes need magnesium ions to coordinate incoming deoxynucleotide triphosphates (dNTPs) during DNA synthesis. This coordination helps ensure the accurate addition of nucleotides to the growing DNA strand.

Polymerase activity: The DNA polymerase used in PCR requires magnesium ions to function optimally. Magnesium ions are essential for the polymerase to bind to the primer-template junction, catalyze the polymerization of nucleotides, and promote the extension of DNA strands.

Question 23

How does MgCl2 contribute to the fidelity and efficiency of the PCR reaction?

Answer 23

MgCl2 helps maintain the fidelity and efficiency of PCR by enabling the DNA polymerase to accurately and efficiently replicate the DNA template. It assists in precise nucleotide addition and promotes successful DNA synthesis.

Question 24

Can the concentration of MgCl2 be adjusted in a PCR reaction?

Answer 24

Yes, the concentration of MgCl2 can be optimized based on the specific requirements of the PCR reaction. The right Mg2+ concentration is crucial for the success of the amplification.

Question 25

Can PCR amplify RNA directly?

Answer 25

No, PCR cannot directly amplify RNA. RNA is first converted into cDNA (complementary DNA), which is then amplified by PCR.

Question 26

What is the purpose of converting RNA to cDNA before amplification?

Answer 26

The conversion of RNA to cDNA allows for the amplification of gene segments without introns, facilitating gene expression analysis and sequencing.

Question 27

What is RT-PCR, and how is it used in molecular biology?

Answer 27

RT-PCR (Reverse Transcription Polymerase Chain Reaction) is used to convert RNA into cDNA, which can then be amplified by PCR. It is employed to study gene expression, quantify mRNA levels, and perform various molecular biology applications.

Question 28

What are introns in the context of genes?

Answer 28

Introns are sequences of nucleotides in a gene that are transcribed but later excised before the gene is translated. They are non-coding regions in the gene’s sequence.

Question 29

How is RT-PCR related to quantitative PCR (qPCR)?

Answer 29

RT-PCR is used to prepare cDNA from RNA, and qPCR (quantitative PCR) is a technique that quantifies the amount of cDNA, allowing the measurement of mRNA levels as an indicator of gene expression.

Question 30

What is the key difference between “end point” PCR and quantitative PCR (qPCR)?

Answer 30

“End point” PCR is assessed after a fixed number of amplification cycles, while qPCR quantifies DNA product after every cycle.

“End point” PCR is the normal PCR that we learned about.

Question 31

What is the advantage of quantitative PCR (qPCR)?

Answer 31

qPCR allows quantification of the initial target DNA copies, as more copies result in earlier detectable product levels.

Question 32

Why is RNA not ideal for PCR?

Answer 32

Because it is pretty unstable (would probably denature upon heating), its single stranded (CANNOT separate it and have two copies on which you can duplicate it)

Question 33

What is the role of SYBR Green in quantitative PCR (qPCR)?

Answer 33

SYBR Green is a fluorescent dye added to the qPCR reaction mix, and it becomes significantly brighter when bound to double-stranded DNA (dsDNA). This allows for the quantification of dsDNA product in the reaction after each PCR cycle.

(increase in dsDNA means increase in fluorescence)

A special thermocycler records the fluorescent emission, enabling the measurement of DNA quantity in qPCR.

Question 34

How is the amount of PCR product measured in qPCR?

Answer 34

The amount of PCR product is measured by observing the level of fluorescence by the SYBR Green dye. More double-stranded products are produced exponentially with each cycle, leading to increased fluorescence in the “exponential phase.”

Question 35

What is the “plateau phase” in qPCR?

Answer 35

After many PCR cycles, the signal reaches a “plateau phase” as one or more reaction components are exhausted. During this phase, the amplification process slows down.

Question 36

What is the cycle threshold (Ct) in qPCR?

Answer 36

The cycle threshold (Ct) is the cycle number at which the threshold level of fluorescence is first surpassed. This is the level of detectability (the point where we can see our product)

When you conduct qPCR, you monitor the fluorescence, and once it crosses a certain threshold, you note the cycle number at which this happens. The Ct value is an important measure in qPCR because it helps assess the starting quantity of the target material.

To control for non-specific DNA amplification, a no template control (NTC) is often added as a separate reaction in the experiment. In the absence of target DNA or cDNA, this sample should not reach the threshold at any cycle.

Question 37

How can you compare the amount of target cDNA in different samples using qPCR?

Answer 37

In qPCR, if you have more of the target cDNA in the initial reaction mix, it will amplify more quickly. As a result, the Ct value (cycle threshold) of the sample with more target cDNA will be lower than the Ct value of the sample with less target cDNA. This one-cycle difference (e.g., shifting from 21 cycles to 20 cycles) is significant because it indicates a higher initial quantity of the target cDNA in the sample. This allows you to quantify and compare the amount of the target cDNA in cells exposed to different treatments or conditions.

Question 38

Samples containing more target mRNA (and thus more target cDNA) will reach the threshold at an ______ cycle.

a) earlier
b) similar
c) later

Answer 38

Samples containing more target mRNA (and thus more target cDNA) will reach the threshold at an EARLIER cycle.

Question 39

If treatment with Drug X increases the CT for Gene Y by 3 cycles, what is the effect of Drug X on the expression of Gene Y?

Decreased Expression by 3x
Increased Expression by 3x
Decreased Expression by 6x
Increased Expression by 6x
Decreased Expression by 8x
Increased Expression by 8x

Answer 39

Decreased expression by 8x

The CT is the number of cycles it takes to visualize this.
If you have less template to begin with, it’s going to take you three cycles more to get to this threshold. 3 cycles is 3 doublings and therefore 2^3 = 8

Question 40

SYBR green binds dsDNA indiscriminately. How can you be certain that the fluorescent signal is coming exclusively from your desired amplification product?

Answer 40

You can use a technique called melt curve analysis. Here’s how it works:

Melt Curve Analysis: This analysis is performed at the end of the qPCR reaction. After the amplification cycles, the temperature is slowly increased. As the temperature rises, the double-stranded DNA (dsDNA) begins to denature, or “melt.”

Specificity Confirmation: The melting temperature (Tm) of dsDNA is influenced by its length and G-C content. By examining the temperature at which the fluorescence decreases during the melt curve analysis, you can confirm the specificity of your amplification.

Single Desired Product: If you have amplified a single desired DNA product, the melt curve should show a sharp decrease in fluorescence at a specific temperature corresponding to the Tm of your target DNA. This indicates that the amplification product is a specific, single product with the expected Tm.

If there are multiple products or contaminants in your reaction, you might see multiple peaks or a broad melting range, indicating the presence of different DNA fragments with different Tm values.

By performing melt curve analysis, you can verify that the observed fluorescence signal is indeed coming from your desired amplification product and not from nonspecific amplification or contaminants. This helps ensure the specificity and reliability of your qPCR results.

Question 41

If treatment with Drug X decreases the CT for Gene Y by 4 cycles, what is the effect of Drug X on the expression of Gene Y?
You can respond once
Decreased Expression by 4x
Increased Expression by 4x
Decreased Expression by 8x
Increased Expression by 8x
Decreased Expression by 16x
Increased Expression by 16x

Answer 41

Increased expression by 16x

need fewer cycles to get to a detectable product. meaning there is more original (2^4 more therefore 16)

Question 42

What is the purpose of a melt curve analysis in quantitative PCR (qPCR)?

Answer 42

Melt curve analysis in qPCR is used to determine the specificity of the amplification product and to detect the presence of non-specific products or contaminants.

Question 43

How does the melt curve of a single, specific PCR product appear?

Answer 43

In the case of a single, specific PCR product, the melt curve exhibits a sharp and distinct decrease in fluorescence at a specific temperature, corresponding to the melting temperature (Tm) of the target DNA.

Question 44

What does a sharp decrease in fluorescence at a specific temperature during melt curve analysis indicate?

Answer 44

A sharp decrease in fluorescence at a specific temperature in the melt curve indicates the melting of the PCR product, confirming the presence of the desired amplification product.

Question 45

What happens during melt curve analysis when there are non-specific products or contaminants in the reaction?

Answer 45

In the presence of non-specific products or contaminants, the melt curve will show distinct decreases in fluorescence at multiple temperatures, each corresponding to the Tm of different DNA products.

Question 46

How does melt curve analysis help distinguish between a specific PCR product and non-specific products or contaminants?

Answer 46

Melt curve analysis allows you to differentiate between a single, specific PCR product (sharp decrease at one temperature) and non-specific products or contaminants (sharp decreases at multiple temperatures), based on the number and temperature of fluorescence decreases.

Question 47

Non-specific products, including primer-dimers and the products of non-specific annealing and amplification, can decrease the efficiency and fidelity of a PCR reaction. You can avoid non-specific products by increasing the stringency of the reaction.

Identify two conditions that could help avoid non-specific products.

Answer 47

You could increase the stringency of the reaction by
1) increasing the annealing temperature or
2) altering the salt concentration - remember, Mg2+ ions are required as a cofactor for DNA polymerase, but too high an Mg2+ concentration can stabilize mismatched DNA-primer base pairing.

Question 48

Reverse transcription (RT)-PCR can be used to amplify ______ that is generated from _____

Answer 48

Reverse transcription (RT)-PCR can be used to amplify cDNA that is generated from RNA

Question 49

What does in vitro DNA amplification reproduce in the context of DNA replication?

A) DNA’s nucleotide sequences
B) The complete cellular environment
C) The building blocks and machinery of DNA replication
D) The genetic code for protein synthesis

Answer 49

C) The building blocks and machinery of DNA replication