Polymerase Chain Reaction Questions and Answers

Q: Amplification of a DNA fragment by PLR requires primers. Which of the following statements are correct for primers ?(i) Primers with hairpin structure formation are not desirable(ii) Primer sequence should not have long runs (\(\gt\)3) of a single nucleotide(iii) GC content of the primer should be less than 20%(iv) Both the forward and reverse primers must have high percentage of base complementarities

Statements (i) and (ii) are correct, so the answer is (i) and (ii).For PCR primers (the paper prints 'PLR'), hairpins are avoided because a primer folded on itself cannot anneal to the template (i). Long runs of one base are avoided because they encourage mispriming and slippage (ii).Statement (iii) is wrong: the recommended GC content is about 40 to 60%, giving a suitable melting temperature, and a GC content below 20% would make the primer bind too weakly. Statement (iv) is wrong as well: forward and reverse primers should not be complementary to each other, or they anneal together and form primer-dimers.

Q: The PCR technique that allows measuring of DNA amplification at each cycle of PCR rather than the end point detection is :

The technique is Real Time PCR.Real time (quantitative) PCR measures the product as it accumulates, cycle by cycle, through a fluorescent signal from a DNA-binding dye or a sequence-specific probe. The cycle at which fluorescence crosses a threshold reflects the starting amount of template.Hot start PCR keeps the polymerase inactive until the first heating step to prevent non-specific priming. Nested PCR uses a second primer pair inside the first product to improve specificity. Reverse transcriptase PCR first copies RNA into cDNA. All three are normally read at the end point, on a gel.

Amplification of a DNA fragment by PLR requires primers. Which of the following statements are correct for primers ?

(i) Primers with hairpin structure formation are not desirable
(ii) Primer sequence should not have long runs (\(\gt\)3) of a single nucleotide
(iii) GC content of the primer should be less than 20%
(iv) Both the forward and reverse primers must have high percentage of base complementarities

A.(i), (ii) and (iii)
B.(ii), (iii) and (iv)
C.(i) and (ii)
D.(iii) and (iv)

Exam Year(s)

MH SET - 2024, SET Exam - 2024,

Answer: C

Statements (i) and (ii) are correct, so the answer is (i) and (ii).

For PCR primers (the paper prints 'PLR'), hairpins are avoided because a primer folded on itself cannot anneal to the template (i). Long runs of one base are avoided because they encourage mispriming and slippage (ii).

Statement (iii) is wrong: the recommended GC content is about 40 to 60%, giving a suitable melting temperature, and a GC content below 20% would make the primer bind too weakly. Statement (iv) is wrong as well: forward and reverse primers should not be complementary to each other, or they anneal together and form primer-dimers.

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The PCR technique that allows measuring of DNA amplification at each cycle of PCR rather than the end point detection is :

A.Hot start PCR
B.Nested PCR
C.Real Time PCR
D.Reverse Transcriptase PCR

Exam Year(s)

MH SET - 2025, SET Exam - 2025,

Answer: C

The technique is Real Time PCR.

Real time (quantitative) PCR measures the product as it accumulates, cycle by cycle, through a fluorescent signal from a DNA-binding dye or a sequence-specific probe. The cycle at which fluorescence crosses a threshold reflects the starting amount of template.

Hot start PCR keeps the polymerase inactive until the first heating step to prevent non-specific priming. Nested PCR uses a second primer pair inside the first product to improve specificity. Reverse transcriptase PCR first copies RNA into cDNA. All three are normally read at the end point, on a gel.

Discuss in Forum

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