Why E. coli DNA Polymerase Cannot Replace Taq Polymerase in PCR
If E. coli DNA polymerase instead of Taq Polymerase is used in a classical PCR-reaction,A researcher will have to
A. Add fresh enzyme after each denaturation step
B. Carry out denaturation step at 50°C instead of 95°C
C. Use different primers
D. Use water bath instead of thermal block
Polymerase Chain Reaction (PCR) is a widely used molecular biology technique to amplify specific DNA sequences. It relies on the use of a heat-stable DNA polymerase enzyme that can withstand high temperatures during the denaturation step. While Taq polymerase is the most commonly used enzyme for PCR, some researchers might wonder whether E. coli DNA polymerase can be used as a substitute. This article explores why E. coli DNA polymerase is not suitable for PCR and the key differences between E. coli polymerase and Taq polymerase.
Correct Answer:
The correct answer is (A) Add fresh enzyme after each denaturation step.
Understanding PCR and the Role of DNA Polymerase
PCR consists of three main steps that are repeated over multiple cycles to amplify DNA:
1. Denaturation:
- The double-stranded DNA is heated to 94°C–98°C to separate the strands.
- This high temperature breaks the hydrogen bonds between complementary base pairs, producing single-stranded DNA templates.
2. Annealing:
- The reaction mixture is cooled to 50°C–65°C to allow primers to bind to complementary sequences on the single-stranded DNA templates.
3. Extension:
- The reaction is heated to 72°C (optimal temperature for Taq polymerase).
- DNA polymerase extends the primers by adding complementary nucleotides to synthesize a new DNA strand.
Why Taq Polymerase is Preferred for PCR
Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, is a heat-stable enzyme that remains active at high temperatures used during PCR.
Key Properties of Taq Polymerase:
1 Heat stability — retains activity even after repeated heating to 95°C.
2 High efficiency in nucleotide incorporation at 72°C.
3 Resistant to denaturation at high temperatures.
Why E. coli DNA Polymerase Cannot Be Used in PCR
E. coli DNA polymerase is not heat-stable and becomes inactivated at high temperatures used during the denaturation step in PCR.
Challenges with E. coli DNA Polymerase:
-
Heat Sensitivity:
- E. coli DNA polymerase denatures at temperatures above 37°C.
- Since the denaturation step in PCR occurs at 95°C, E. coli DNA polymerase will lose activity after the first cycle.
-
Repeated Addition of Enzyme:
- If E. coli DNA polymerase is used, the researcher would need to add fresh enzyme after each denaturation step.
- This increases the cost, complexity, and chances of contamination.
-
Low Extension Temperature:
- E. coli DNA polymerase functions optimally at 37°C.
- It cannot efficiently extend DNA strands at 72°C, the standard extension temperature for PCR.
-
Thermal Inactivation:
- The high denaturation temperature inactivates E. coli DNA polymerase.
- Unlike Taq polymerase, it cannot withstand high thermal cycling.
Explanation of Why Other Options Are Incorrect:
(B) Carry out denaturation step at 50°C instead of 95°C
❌ Incorrect — The denaturation step requires temperatures above 90°C to break hydrogen bonds between DNA strands. Lowering the temperature to 50°C would prevent proper strand separation, leading to PCR failure.
(C) Use different primers
❌ Incorrect — Primers are designed based on the DNA target sequence, not the type of polymerase used. Therefore, changing primers would not solve the heat sensitivity problem of E. coli polymerase.
(D) Use a water bath instead of a thermal block
❌ Incorrect — A thermal cycler provides precise temperature control for denaturation, annealing, and extension. Using a water bath would not allow accurate temperature control required for PCR.
Comparison Between Taq Polymerase and E. coli DNA Polymerase
| Feature | Taq Polymerase | E. coli DNA Polymerase |
|---|---|---|
| Source | Thermus aquaticus | Escherichia coli |
| Optimal Temperature | 72°C | 37°C |
| Heat Stability | Stable at 95°C | Denatures at 40°C |
| Need for Fresh Enzyme | No | Yes |
| Efficiency in PCR | High | Low |
Why Taq Polymerase is Ideal for PCR
1 High thermal stability enables multiple PCR cycles without loss of activity.
2 Efficient extension at high temperatures improves yield and specificity.
3 Reduced contamination risk due to stable enzyme activity.
4 Suitable for high-throughput PCR and automated systems.
Applications of Taq Polymerase in PCR
-
Genetic Research:
- Gene cloning and sequencing.
- Mutation analysis and genotyping.
-
Clinical Diagnostics:
- Detection of genetic disorders.
- Identification of pathogens (e.g., SARS-CoV-2).
-
Forensic Science:
- DNA fingerprinting for criminal investigations.
-
Agriculture:
- Detection of genetically modified organisms (GMOs).
Challenges of Using E. coli DNA Polymerase in PCR
- High Cost: Repeated enzyme addition increases the overall cost of PCR.
- Contamination Risk: Frequent handling increases the risk of contamination.
- Low Efficiency: E. coli polymerase does not work efficiently at high temperatures.
- Inactivation: Rapid inactivation at high temperatures limits its utility in PCR.
How to Improve PCR Performance
1 Use Taq polymerase or other thermostable enzymes (e.g., Pfu polymerase) for improved fidelity.
2 Optimize primer design to improve specificity and yield.
3 Maintain consistent cycling conditions using a thermal cycler.
4 Minimize contamination by using sterile reagents and pipette tips.
Conclusion
E. coli DNA polymerase is not suitable for PCR because it is heat-sensitive and becomes inactivated at high temperatures used during denaturation. The correct answer is (A) Add fresh enzyme after each denaturation step since E. coli polymerase would need to be replenished after every PCR cycle. Taq polymerase, on the other hand, remains stable at high temperatures and efficiently extends DNA strands, making it the preferred enzyme for PCR. Understanding the properties of DNA polymerases is crucial for successful PCR amplification and accurate DNA analysis.
68 Comments
Nisha
March 17, 2025Done
Akshay mahawar
March 17, 2025Done 👍
Arushi
March 17, 2025Nice explanation 👍
Suman bhakar
March 20, 2025👍
Parul
March 23, 2025Okay sir done.
Ujjwal
March 26, 2025Done sir
Mohd juber Ali
August 24, 2025E coli. Dna polymerase is not heat stable enzyme
Denaturation step temperature= 95° High.
Tmp.
So E. coli high temperature pe inactivated ho jata h so after the denaturation step add fresh enzyme
On other hand taq polymerase is heat stable (high temperature 95°)
Mansukh Kapoor
August 24, 2025The correct answer is option 1st
Add fresh enzyme after each denaturation step because E.coli doesn’t tolerate high temperature
Anurag Giri
August 24, 2025Ecoli dna polymerase not a heat stable Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, is a heat-stable enzyme that remains active at high temperatures used during PCR
Khushi Agarwal
August 24, 2025Correct answer is a
Add fresh enzyme after each denaturation step
Bcz e coli degrade after denaturation
Avni
August 24, 2025The correct answer is (A) Add fresh enzyme after each denaturation step because it denatures at high temperature
Soniya Shekhawat
August 24, 2025Add a fresh enzyme after the denaturation step because DNA polymerase is heater sensitive so it is be degrade easily so after denaturation adding the fresh enzyme for continueously processof PCR instead taq polymerase thermophilic and acidophilic tolerate at Harsh condition. So 1 st is right.
Neelam Sharma
August 24, 2025Add fresh enzyme after each denaturation step because e coli DNA polymerase enzyme not heat stable
Denaturation at 40° C and low efficiency in PCR
Sheikh Aleen
August 24, 2025Add fresh enzyme after each denaturation step because it denatures at temperatures above 37°C.
Roopal Sharma
August 24, 2025Option A adding fresh enzyme after each denaturation step.
Khushi Pareek
August 24, 2025Ecoli DNA polymerase is not thermostatic hence have to add fresh polymerase after denaturation step
Divya rani
August 24, 2025B is right because e.coli DNA polymerase is not heat stable or thermostable so we have to change the enzyme after Every denaturation step in PCR but Taq polymerase is a thermostable enzyme That’s why it is more suitable for PCR.
Priyanka Choudhary
August 24, 2025Correct answer is option b ,Add fresh enzyme after each denaturation step because ecoli degrade after denaturaion
Dipti Sharma
August 24, 2025Add fresh enzyme after each denaturation step bcz ecoli dna polymerase is not stable at high temperature unlike taq polymerase
anjani sharma
August 24, 2025E. coli DNA polymerase is not heat-stable and becomes inactivated at high temperatures
So we have to add fresh e.coli at every step of denaturation
Neha Yadav
August 24, 2025E.coli DNA polymerase is not heat stable and become inactivated at high temperature used during the denaturation step in pcr so add fresh enzyme after each denaturation step
MOHIT AKHAND
August 24, 2025Done sir ✅
Tanvi Panwar
August 24, 2025Add fresh denaturation enzymes after each cycle.
Heena Mahlawat
August 24, 2025Add fresh enzyme after denaturation step
Arushi Saini
August 24, 2025I. Denaturation Ecoli dna ploymerase will become inactive so we have to add fresh enyme after every denaturation step
Aakanksha Sharma
August 24, 2025Add fresh enzyme after each denaturation step
Bharti Yadav
August 24, 2025Add fresh enzyme after each denaturation step. because E.coli dna polymerase cannot stable at high temperature
Sneha Kumawat
August 24, 2025E.coli DNA polymerase is not heat stable enzyme it’s denatures at 40 degree C. Need for fresh enzyme
Ayush Dubey
August 25, 2025Add fresh enzyme after each denaturation step.
Dharmpal Swami
August 25, 2025Use Taq polymerase then add fresh denaturation enzyme after each cycle
Mitali saini
August 25, 2025The correct answer is (A) Add fresh enzyme after each denaturation step.
Anjali
August 25, 2025Ans is a
Surbhi Rajawat
August 25, 2025Because DNA polymerase of Ecoli is not stable at high temperature therefore we’ll have to add a new sample after each denaturation
shruti sharma
August 25, 2025Add fresh enzyme after each denaturation step
Vanshika Sharma
August 26, 2025Add fresh enzyme after every denaturation step
Rishita
August 26, 2025Done
Aafreen Khan
August 26, 2025Add fresh enzyme after each denaturation step because DNA polymerase of E.Coli doesn’t stable at High temperature thats why add fresh enzyme or sample after each denaturation
Minal Sethi
August 26, 2025Fresh enzyme needs to be added after every denaturation step because the existing enzyme will be denatured at high temperature
Shivani
August 26, 2025Add fresh enzyme after each denaturation step because e coli. Is not heat stable and became unstable at high temp.
Aman Choudhary
August 26, 2025E.coli polymerase doesn’t tolerate heat therefore thermophilic bacterium Thermus aquaticus, is a heat-stable enzyme that remains active at high temperatures
Seema
August 26, 2025A)add fresh enzymes after each denaturation step becouse ecoli is not heat stable and become unstable after each denaturation step.
Alec
August 26, 2025add fresh enzyme after each denaturation step because ecoli is not thermostable.
Surendra Doodi
August 26, 2025Add fresh enzyme after each denaturation step. Because E-coli is not thermostable
Priya dhakad
August 27, 2025Add fresh enzyme after each denaturation step .
Rakesh Dhaka
August 27, 2025Correct answer is a
Ecoli dna polymerase not a heat stable Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, is a heat-stable enzyme that remains active at high temperatures used during PCR
Sakshi Kanwar
August 27, 2025E.coli DNA polymerase is not heat stable cannot work at denaturation step again and again so need to add enzyme at each step of denaturation
Mohini
August 28, 2025A) Add fresh enzyme after each denaturation step.
Kanica Sunwalka
August 28, 2025beoz dna pol of e.coli is not heat stable
becomes inactivated during high temp during denaturation step in pcr
Deepika Sheoran
August 28, 2025Option A is correct
Add Fresh enzyme after each Denaturation step because E.coli. doesn’t tolerate High temperature. That remains Active at High temperature used during PCR..
Khushi Vaishnav
August 28, 2025Add fresh enzyme after each denaturation step
Devika
August 29, 2025Option A add fresh enzyme after each denaturation step
Muskan singodiya
August 29, 2025A
Add a fresh enzyme after each denaturation step
Simran Saini
August 29, 2025Add fresh enzyme after each denaturation step.
Anisha Beniwal
August 29, 2025Add fresh enzyme after each denaturation step
Neeraj Sharma
August 29, 2025After denaturation the polymerase enzyme will degrade as it is not thermostable so the scientist have to add a new fresh enzyme every time after denaturation
Asha Gurzzar
August 30, 2025After each step of denaturation pe fresh enzyme use krna pdega
Aartii sharma
August 30, 2025After each step of denaturation we have to use fresh enzyme
Khushi Singh
August 30, 2025E.colk doesn’t tolerate high temperature
Kajal
August 30, 2025If dna polymerase is added instead of taq polymerase then fresh enzyme addition is required after each step of dna denaturation
Sonam Saini
August 30, 2025If DNA polymer is added interested of tag polymer is the fresh enzyme addition is required after each step of DNA denaturation
Pooja Sharma Sharma
August 31, 2025If E. coli DNA polymerase instead of Taq Polymerase is used in a classical PCR-reaction, A researcher will have to add fresh enzyme after each denaturation step
Kirti Agarwal
August 31, 2025Add fresh enzyme and denaturation step
Palak Sharma
August 31, 2025Add fresh enzyme after each denaturation step .
Kajal
August 31, 2025correct answer is (A) Add fresh enzyme after each denaturation step
Sakshi yadav
August 31, 2025Add fresh enzyme after each denaturation step
Konika Naval
August 31, 2025Add fresh enzyme after each denaturation step.
Khushi Mehra
September 8, 2025After each denaturation step , a researcher have to add fresh enzyme because DNA polymerase does not tolerate high temperature.
Muskan Yadav
September 17, 2025Add fresh enzyme after each denaturation step.