One way to detect and measure accurately the amount of RNA in a tissue sample is by RT-PCR (reverse transcriptase-polymerase chain reaction). RT-PCR uses a reaction mixture containing: - the sample for testing - reverse transcriptase - DNA nucleotides - primers - DNA polymerase - fluorescent dye. The principle behind this method is shown in Figure 8. **Figure 8** Amount of DNA amplified by PCR depends on the amount of RNA in the sample. The dye only fluoresces when bound to DNA. The intensity of the fluorescent light emitted increases as the PCR products accumulate. **8.1** Explain the role of reverse transcriptase in RT-PCR. **8.2** Explain the role of DNA polymerase in RT-PCR. **8.3** Any DNA in the sample is hydrolysed by enzymes before the sample is added to the reaction mixture. Explain why. **8.4** Figure 9 shows the results from using RT-PCR to detect RNA in two different samples, A and B. **Figure 9** A quantitative comparison can be made of the amount of RNA in samples A and B. This involves determining the number of cycles required to reach 50% maximum concentration of DNA (C). The amount of RNA in a sample can be measured as: $rac{1}{C}$. Use this information to calculate the ratio for RNA content in sample A: RNA content in sample B. **8.5** Suggest one reason why DNA replication stops in the polymerase chain reaction. **8.6** Scientists have used the RT-PCR method to detect the presence of different RNA viruses in patients suffering from respiratory diseases. Explain why. The scientists produced a variety of primers for this procedure.

A-Level AQA Biology Regulation of Gene Expression: One way to detect and measure ac

One way to detect and measure accurately the amount of RNA in a tissue sample is by RT-PCR (reverse transcriptase-polymerase chain reaction) - AQA - A-Level Biology - Question 8 - 2017 - Paper 2

Question 8

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One way to detect and measure accurately the amount of RNA in a tissue sample is by RT-PCR (reverse transcriptase-polymerase chain reaction). RT-PCR uses a reaction... show full transcript

Worked Solution & Example Answer:One way to detect and measure accurately the amount of RNA in a tissue sample is by RT-PCR (reverse transcriptase-polymerase chain reaction) - AQA - A-Level Biology - Question 8 - 2017 - Paper 2

Step 1

Explain the role of reverse transcriptase in RT-PCR.

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Answer

Reverse transcriptase is an enzyme that synthesizes complementary DNA (cDNA) from a template of messenger RNA (mRNA). This conversion is essential for amplifying RNA sequences in the RT-PCR process.

Step 2

Explain the role of DNA polymerase in RT-PCR.

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Answer

DNA polymerase is responsible for synthesizing new DNA strands by adding nucleotides to the growing cDNA during the PCR amplification process, thus allowing the exponential increase of DNA.

Step 3

Explain why any DNA in the sample is hydrolyzed by enzymes before the sample is added to the reaction mixture.

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Answer

DNA is hydrolyzed to prevent it from being amplified during RT-PCR, as the presence of DNA would compete with the target RNA for amplification and could lead to inaccurate results.

Step 4

Use this information to calculate the ratio for RNA content in sample A: RNA content in sample B.

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Answer

To calculate the ratio of RNA content, identify the cycle numbers where the fluorescence levels reach 50% of the maximum for samples A and B from Figure 9. Let's assume A required 15 cycles and B required 20 cycles. Thus, the RNA content ratio is calculated as:

$ext{Ratio} = rac{1 / C_A}{1 / C_B} = rac{C_B}{C_A} = rac{20}{15} = rac{4}{3} ext{ or 1.33:1}$

Step 5

Suggest one reason why DNA replication stops in the polymerase chain reaction.

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Answer

DNA replication stops due to a limited amount of primers or nucleotides, which become exhausted as the reaction progresses.

Step 6

Explain why the scientists produced a variety of primers for this procedure.

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Answer

The scientists produced a variety of primers to ensure specificity for different RNA viruses, allowing them to detect multiple viral targets in patients suffering from respiratory diseases.

One way to detect and measure accurately the amount of RNA in a tissue sample is by RT-PCR (reverse transcriptase-polymerase chain reaction) - AQA - A-Level Biology - Question 8 - 2017 - Paper 2

Worked Solution & Example Answer:One way to detect and measure accurately the amount of RNA in a tissue sample is by RT-PCR (reverse transcriptase-polymerase chain reaction) - AQA - A-Level Biology - Question 8 - 2017 - Paper 2

Explain the role of reverse transcriptase in RT-PCR.

Explain the role of DNA polymerase in RT-PCR.

Explain why any DNA in the sample is hydrolyzed by enzymes before the sample is added to the reaction mixture.

Use this information to calculate the ratio for RNA content in sample A: RNA content in sample B.

Suggest one reason why DNA replication stops in the polymerase chain reaction.

Explain why the scientists produced a variety of primers for this procedure.

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