ASSIGNMENT A qPCR analysis was performed on three different samples: For every q

Question

ASSIGNMENT A qPCR analysis was performed on three different samples:

For every question (Q1- Q6) below compare the statements (A and B) and select the correct answer (1, 2 or 3):

Answer options:

1. A is greater than B

2. B is greater than A

3. The two are equal or very nearly equal

Q1: Answer__________

A. The number of free primer molecules in sample 3 after cycle 3

B. The number of free primer molecules in sample 3 after cycle 10

Q2: Answer__________

A. The number of free primer molecules in sample 1 after cycle 5

B. The number of free primer molecules in sample 3 after cycle 5

Q3: Answer__________

A. The number of free probe molecules in sample 1 after cycle 5

B. The number of free probe molecules in sample 3 after cycle 5

Q4: Answer__________

A. The number of free probe molecules in sample 1 after cycle 20

B. The number of free probe molecules in sample 2 after cycle 20

Q5: Answer__________

A. The number of amplified target sequence fragments in sample 1 after cycle 20

B. The number of amplified target sequence fragments in sample 2 after cycle 20

Q6: Answer_________

A. The number of amplified target sequence fragments in sample 1 after cycle 45

B. The number of amplified target sequence fragments in sample 2 after cycle 45

Explanation / Answer

Solution for Q1

In a PCR, primer molecules will be higher in initial cycles of a reaction. So, the number of free primers after cycle 3 will be higher than the primers available after cycle 10 in sample 3. So, option 1 - A is greater than B is correct.

Solution for Q2

Since both the samples (Sample 1 and Sample 3) are in the same cycle, the number of free primers will be same. So, option 3 - The two are equal or nearly equal is correct.

Solution for Q3

Sample 1 has started to show fluorescent signal after cycle 5. Hence, the number of free probe molecules after cycle 5 must reduce. Sample 3 has not shown any signal after cycle 5. Hence the number of free probe molecules after cycle 5 must be same as the earlier cycles.

So, the correct answer to this question is option 2 - B is greater than A

Solution for Q4

In real-time PCR, the availability of free probe molecules will be higher in samples with lower Ct values. A Ct value defines the number of cycles required by a sample to cross the threshold signal. As per the given figure, sample 1 has lower Ct value than the sample 2 and hence the availability of free probe molecules are higher for sample 1. So, after cycle 20, sample 1 will have higher probe molecules than the sample 2 at the same cycle.

The correct answer to this question is Option 1 - A is greater than B.

Note: In the previous question we are not considering the Ct values. This is because cycles in both samples have not crossed the threshold point.

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