Reason for Specific Point Mutation in PCR Products
Why specific point mutation appears in all the PCR products during the amplification of a genomic DNA segment of interest. Where the primers are designed according to genomic database.
(a) The presence of a mutation in the primer itself
(b) The sequence available in the database is wrong
(c) Both (a) and (b) are correct
(d) The polymerase enzyme does not have proof-reading activity
The correct answer is (d) The polymerase enzyme does not have proofreading activity.
✅ Correct Answer: (d) The polymerase enzyme does not have proofreading activity
Explanation
PCR (Polymerase Chain Reaction) relies on DNA polymerase to amplify a specific DNA segment. If specific point mutations consistently appear in PCR products, it indicates that the DNA polymerase used lacks proofreading activity.
🔬 Why Lack of Proofreading Causes Mutation
-
Proofreading Function of DNA Polymerase:
- High-fidelity DNA polymerases (e.g., Pfu polymerase) have 3′ → 5′ exonuclease activity that allows them to detect and correct misincorporated nucleotides during amplification.
- If the polymerase lacks this proofreading activity (e.g., Taq polymerase), it cannot detect and correct errors, leading to misincorporation of nucleotides and consistent point mutations.
-
Propagation of Error:
- Once a mutation is introduced in the early PCR cycles, it becomes part of the template for subsequent cycles.
- This leads to the consistent appearance of the same point mutation in all PCR products.
-
High Error Rate of Non-Proofreading Polymerases:
- Non-proofreading polymerases, such as Taq polymerase, have an error rate of approximately 1 error per 10,000 bases incorporated.
- This increases the likelihood of consistent point mutations during multiple rounds of amplification.
✅ Example of PCR Error Due to Lack of Proofreading:
- Template:
- Original sequence: 5′ – ATGCGA – 3′
- Error during first cycle (due to lack of proofreading):
- Mutated sequence: 5′ – ATGTGA – 3′ (G instead of C)
- Mutation propagates in subsequent cycles, leading to consistent incorporation of incorrect base pairs.
Why Other Options Are Incorrect
| Option | Explanation | Correct/Incorrect |
|---|---|---|
| (a) The presence of a mutation in the primer itself | A mutation in the primer would introduce a consistent error, but it would be primer-dependent rather than polymerase-dependent. | ❌ Incorrect |
| (b) The sequence available in the database is wrong | Incorrect database sequence would lead to mismatched primer binding, but not consistent point mutations. | ❌ Incorrect |
| (c) Both (a) and (b) are correct | Incorrect primer or database sequence would cause amplification issues, but not systematic mutation. | ❌ Incorrect |
| (d) The polymerase enzyme does not have proofreading activity | Lack of proofreading activity leads to consistent and reproducible point mutations. | ✅ Correct |
How to Confirm the Cause of Mutation
✅ 1. Use High-Fidelity Polymerase:
- Enzymes like Pfu polymerase and Phusion polymerase have 3′ → 5′ exonuclease activity and low error rates.
✅ 2. Sequence the PCR Product:
- Direct sequencing can confirm whether the mutation arises from polymerase error or template error.
✅ 3. Perform Control Reactions:
- Repeat PCR with different polymerases (proofreading and non-proofreading) to confirm the source of mutation.
✅ 4. Optimize PCR Conditions:
- Use a lower number of cycles to reduce the error rate.
- Optimize annealing temperature to improve primer specificity.
Strategies to Avoid Mutation Due to Polymerase Errors
✅ 1. Use High-Fidelity Polymerases:
- Examples:
- Pfu polymerase
- Phusion polymerase
- Q5 polymerase
✅ 2. Adjust PCR Conditions:
- Reduce cycle number to minimize error accumulation.
- Optimize magnesium concentration and extension time.
✅ 3. Use Hot-Start Polymerase:
- Prevents non-specific binding and extension at lower temperatures.
✅ 4. Perform Cloning and Sequencing:
- Sequence multiple clones to confirm the true sequence.
- Identify and eliminate mutants.
Applications of High-Fidelity PCR
✅ 1. Site-Directed Mutagenesis
- High-fidelity polymerases are essential for introducing targeted mutations.
✅ 2. Cloning and Gene Expression
- Accurate amplification ensures that the gene of interest is not altered.
✅ 3. Next-Generation Sequencing (NGS)
- High-fidelity amplification is crucial for accurate sequence representation.
✅ 4. Disease Diagnostics
- Detecting mutations in clinical samples requires high accuracy.
Summary
- Consistent point mutations in PCR products are most often due to the use of non-proofreading DNA polymerases (e.g., Taq polymerase).
- High-fidelity polymerases with 3′ → 5′ exonuclease activity can prevent such errors.
- Careful optimization of PCR conditions and proper enzyme selection are key to avoiding point mutations.
72 Comments
Akshay mahawar
March 17, 2025Done 👍
Ujjwal
March 17, 2025Done sir
Nisha
March 17, 2025Done
Mahima Sharma
August 24, 2025Polimerase have not proof reading activity
Arushi
March 17, 2025👍👍
Suman bhakar
March 17, 2025👍👍
Anmol
March 17, 2025Done 👍🏻
Parul
March 23, 2025Done sir.
Devika
August 26, 2025The polymerase enzyme does not have proof reading activity
Roopal Sharma
August 24, 2025Lack of proof readimg led to pint mutations.
Neha Yadav
August 24, 2025Specific point mutations in all pcr as the dna polymerase enzyme does not have the proof reading activity
Soniya Shekhawat
August 24, 2025Lack of proof reading activity of polymerase lead to be a point mutation.
Mohd juber Ali
August 24, 2025If specific point mutation appear in PCR product that means dna poly.used lack proof reading activity
Lack proof reading activity (3′ to 5′ exonuclease) can not detect and correct error
Khushi Pareek
August 24, 2025Taq polymerase lacks proofreading activity
Anurag Giri
August 24, 2025In PCR DNA polymerase to amplify a specific DNA segment. If specific point mutations consistently appear in PCR products, it indicates that the DNA polymerase used lacks proofreading activity.
Sakshi yadav
August 24, 2025Lack proof reading activity eg taq polymerase
Avni
August 24, 2025The polymerase enzyme does not have proofreading activity
Mansukh Kapoor
August 24, 2025The correct answer is option 4th
The polymerase enzyme does not have proofreading activity(3’to5′ exonuclease activity)
Khushi Agarwal
August 24, 2025(c) Both (a) and (b) are correct
Mutation ka reason ya to primer ki khud ki sequence galti hai, ya database sequence galat tha
Dono condition me mutation har product me same position par rahega, bcz primer har round me us wrong base ko introduce karta rahega
Aafreen Khan
August 24, 2025The polymerase enzyme does not have proof-reading activity bcoz specific point mutations appear in PCR it indicates that the DNA Polymerase lacks proofreading activity
Surbhi Rajawat
August 24, 2025Primer designing is prone to mutation because unlike our body cells, the polymerase that we use does not have proof reading activity
Yashika Choudhary
August 24, 2025Continious mutation in pcr amplification says that polymerase lack proof reading activity
Dipti Sharma
August 24, 2025Lack of proofreading activity in taq polymerase leads to consistent point mutations.
Priyanka Choudhary
August 24, 2025The polymerase enzyme does not have proofreading activity is right answer d,(3’to5′ exonucleases)
Sneha Kumawat
August 24, 2025The polymerse enzyme does not have proof reading activity
Santosh Saini
August 24, 2025Lack of proofreading activity (3′ to 5’exonuclease) leads to consistent and reproducible point mutations in PCR, so we can not detect
Tanvi Panwar
August 24, 2025The polymerase enzyme does not have proof reading activity.
anjani sharma
August 24, 2025Answer 4
Lack of proofreading activity leads to point mutations
Aman Choudhary
August 24, 2025Option D is correct
The polymerase enzyme does not have proof-reading activity
anjani sharma
August 24, 2025Answer 4
Lack of proofreading activity leads to point mutations
MOHIT AKHAND
August 24, 2025Done sir ✅
Neelam Sharma
August 24, 2025The polymerase enzyme does not have proofreading activity.
Bhawna Choudhary
August 24, 2025Polymerase enzyme does not have proof reading activity
Ayush Dubey
August 24, 2025The polymerase enzyme does not have proofreading activity
Monika jangid
August 24, 2025Ans – d polymerase doesnt have proof reading activity
Priyanka Verma
August 24, 2025Answer D is correct – polymerase enzyme does not have proof reading activity.
Lack of proofreading activity leads to point mutations
Bharti Yadav
August 24, 2025The polymerase enzyme does not have proofreading activity.
Falguni Prakash
August 24, 2025D
Varsha Tatla
August 24, 2025In PCR,dna lack proofreading activity
Divya rani
August 25, 2025Lack of proof reading activity in polymerase enzyme which leads to consistent and reproducible point mutation.
shruti sharma
August 25, 2025The pol enzyme does not have proof reading activity
Priti khandal
August 25, 2025The polymerase enzyme does not have proof reading activity
Minal Sethi
August 25, 2025Taq Polymerase do not have proof reading activity
Anjana sharma
August 25, 2025Option d is correct dna polymerase and taq polymerase both don’t have proof reading mechanism due to which error possibility are high
Vanshika Sharma
August 25, 2025Lack of proof reading activity will leads to repeat mutation every time
Surendra Doodi
August 25, 2025The some polymerase enzyme does not have proofreading activity
Bhavana kankhedia
August 26, 2025The polymerase enzyme does not have proofreading activity
Manisha
August 26, 2025Lack of proofreading activity leads to consistent and reproducible point mutations.
Shivani
August 26, 2025(d) The polymerase enzyme does not have proofreading activity.
Rishita
August 26, 2025Done sir 👍🏻
Alec
August 26, 2025The polymerase enzyme does not have proof reading ability.
Pallavi Ghangas
August 26, 2025Polymers lack proof reading enzyme due to which point mutations occur in PCR
Simran Saini
August 26, 2025The Polymerase enzyme does not have proofreading activity.
Nilofar Khan
August 27, 2025Correct answer is d
The polymerase enzyme does not have proof-reading activity
Mohini
August 27, 2025Option D is correct. The polymerase lacks proofreading activity.
Kanica Sunwalka
August 28, 2025option d
Kajal
August 28, 2025Specific point mutations occurs in all PCR the reason behind this is that the polymerase does not have proof reading activity
Muskan singodiya
August 28, 2025The polymerase enzyme does not have proofreading
Deepika sheoran
August 28, 2025Option D is correct
The polymerase enzyme does not have proof reading Activity..
Neeraj Sharma
August 28, 2025Polymerase enzyme does not have proof reading activity
Anisha Beniwal
August 28, 2025Option D is correct
Parul Yadav
August 29, 2025Specific point mutation appears in all PCR products during amplification of segment of genomic DNA because Taq Polymerase lacks proof reading activity.
Asha Gurzzar
August 30, 2025D is correct as polymerase lack proof reading activity
Khushi Singh
August 30, 2025Polymerase has do not have proof reading
Sonam Saini
August 30, 2025Polymerase enzymes do not have full proofreading activity
Kirti Agarwal
August 31, 2025Lack proofreading activity
Kajal
August 31, 2025Correct Answer is (d) The polymerase enzyme does not have proofreading activity
Aartii sharma
August 31, 2025Polymerase enzyme does not have proofreading activity
Konika Naval
August 31, 2025Option d 👍
Meenakshi Choudhary
September 3, 2025The polymerase enzyme does not have proof-reading activity
Yashika Choudhary
September 6, 2025Sopecific point mutstiom means dna polymerase lack proff reading activity
Muskan Yadav
September 8, 2025The polymerase enzyme does not have proofreading activity