Reason for PCR Failure Due to Incorrect Annealing Temperature
A scholar set up a PCR to copy a specific gene promoter using primers based on genomic database and a new thermostable polymerase with 5′ —- 3′ exonuclease activity. But he could not get amplified DNA because
(a) Annealing temperature was not right
(b) Genomic DNA was contaminated
(c) Proof-reading function was lacking
(d) One or more nucleotides were lacking
The correct answer is (a) Annealing temperature was not right.
✅ Correct Answer: (a) Annealing temperature was not right
Explanation
PCR (Polymerase Chain Reaction) involves three key steps:
- Denaturation – Separation of DNA strands at high temperature (~94–98°C).
- Annealing – Binding of primers to the template strand at an optimized temperature (50–65°C).
- Extension – DNA polymerase extends the primers to synthesize the new DNA strand at ~72°C.
Why Annealing Temperature Matters
-
Too High Annealing Temperature:
- Primers may fail to bind to the template.
- Results in no amplification or very low yield.
-
Too Low Annealing Temperature:
- Non-specific binding of primers to mismatched sequences.
- Results in non-specific amplification or multiple bands.
-
Optimized Annealing Temperature:
- Ideal temperature is calculated using the melting temperature (Tm) of the primers.
- Formula:
Tm=4(G+C)+2(A+T)Tm = 4(G + C) + 2(A + T) - Annealing temperature is typically set at 5°C below Tm.
Why Other Options Are Incorrect
| Option | Explanation | Correct/Incorrect |
|---|---|---|
| (a) Annealing temperature was not right | Incorrect annealing temperature prevents proper primer binding, leading to no amplification. | ✅ Correct |
| (b) Genomic DNA was contaminated | Contamination may lead to additional bands or poor specificity but does not prevent amplification altogether. | ❌ Incorrect |
| (c) Proof-reading function was lacking | Lack of proofreading reduces accuracy but does not prevent amplification. | ❌ Incorrect |
| (d) One or more nucleotides were lacking | Absence of nucleotides reduces yield but does not prevent initial binding and extension. | ❌ Incorrect |
How to Optimize Annealing Temperature
- Calculate Tm using the primer sequence.
- Start with a gradient PCR to determine the ideal temperature.
- Use an annealing temperature approximately 5°C lower than the primer Tm.
- Adjust for:
- GC content – Higher GC content → Higher annealing temperature
- Primer length – Longer primers → Higher annealing temperature
- Salt concentration – Higher salt → Higher annealing temperature
Effects of Incorrect Annealing Temperature
| Condition | Effect |
|---|---|
| High Annealing Temperature | No binding of primers → No amplification |
| Low Annealing Temperature | Non-specific binding → Multiple bands |
| Optimized Annealing Temperature | Specific amplification of target DNA |
Example:
If the primer sequence is:
5′-GACCTGTGGAAGC-3′
- GC content = 7
- AT content = 6
Using the formula:
Tm = 4(7) + 2(6) = 28 + 12 = 40°C
Set the annealing temperature at ~35°C for successful primer binding.
Solutions to Improve PCR Success
✅ Use gradient PCR to optimize annealing temperature.
✅ Adjust magnesium concentration for better primer-template interaction.
✅ Increase the length of primers to increase specificity.
✅ Ensure correct Tm calculation for both primers.
Applications of PCR-Based Gene Amplification
✅ 1. Cloning
Amplification of gene promoters for insertion into vectors.
✅ 2. Gene Expression Studies
PCR-based quantification of gene promoter activity.
✅ 3. Mutagenesis
Site-directed mutagenesis using modified primers.
✅ 4. Genotyping
Identification of genetic variations using PCR.
Summary
- PCR failure occurred because the annealing temperature was not optimized.
- Incorrect temperature prevents proper primer binding, leading to no DNA amplification.
- Solution: Adjust the annealing temperature using gradient PCR and correct Tm calculation.
85 Comments
Akshay mahawar
March 17, 2025Done 👍
Mahima Sharma
August 24, 2025High temperature so no binding
Suman bhakar
March 17, 2025💯
Anmol
March 17, 2025Done
Parul
March 23, 2025Done sir.
Ujjwal
March 24, 2025✔️✔️
Anita Choudhary
April 19, 2025Done
Diksha Chhipa
August 23, 2025Annealing temp must be optimize
Khushi Pareek
August 23, 2025Annealing temp varies acc to the tm
Hence ans is Annealing temperature was not right
Roopalsharma
August 24, 2025Annealing temperature should be optimized because at high temperature primers didn’t bind and at low temperature non specific dna led to mismatching . All other options are incorrect because contaminated dna can’t stop amplification it led to low specificity
Neha Yadav
August 24, 2025Reason for PCR failure : Annealing temp was not right due to which may be primer not bind to its complementary sequence to initiate dna synthesis so couldn’t get amplified dna
Soniya Shekhawat
August 24, 2025If annealing temperature is very high them primer is not bind to DNA and if annealing temperature is the low temperature them non specific binding or producing the multiple band so specific annealing temperature is be required for the specific DNA target DNA and other option is be held is incorrect because other option is occur so there is a low specificity but not a but not a stop the amplification of DNA.
Santosh Saini
August 24, 2025Annealing temperature was not right so primers not bind to its complementary sequence, primer binds only 50-68° temp. , so not amplified to DNA
Mohd juber Ali
August 24, 2025High temperature per primer bind nahi hote na hi low temperatures pe bind hote
Melting tmp (optimised tmp) pe annealing hogi( primer bind )
Priya dhakad
August 24, 2025Incorrect annealing temperature prevents proper primer binding, leading to no amplification.so option A is correct
Anurag Giri
August 24, 2025Ans 1
PCR failure occurred because the annealing temperature was not optimized.
Incorrect temperature prevents proper primer binding, leading to no DNA amplification.
HIMANI FAUJDAR
August 24, 2025Ans Annealing,In PCR three main steps involved i.e Denaturation ,Annealing and Extension So if Annealing temperature is too high or too low it can prevent the binding of primers and cause failure in PCR.
Aafreen Khan
August 24, 2025Annealing temperature was not right
At too high temperature Primers fail to bind and no amplification.
At low temperature also binding of non-specific primers and result has multiple bands
Sakshi yadav
August 24, 2025Annealing temp.was not right so the primers are not bind at specific site and no band found
Avni
August 24, 2025The correct answer is (a) Annealing temperature was not right.
Incorrect annealing temperature prevents proper primer binding, leading to no amplification.
Arushi Saini
August 24, 2025Annealing temprature was not right
Mansukh Kapoor
August 24, 2025The correct answer is option 1st
Annealing temp was not right
karishma don
August 24, 2025due to wrong annealing temperature, if high annealing temp then primers dont bind and amplify and too low annealing temp causes non specific primers to bind
Khushi Agarwal
August 24, 2025Option a is correct
Bcz annealing tem. Prevents proper primer bind , leading to no amplify
Surbhi Rajawat
August 24, 2025Getting no bands of amplified DNA points towards improper annealing temperature. Which is a crucial step od PCR and has to be monitored very carefully
Aakanksha Sharma
August 24, 2025Annealing temperature was not right
Dharmpal Swami
August 24, 2025Optimise Anneling temprature -specific amplification for target dna
Yashika Choudhary
August 24, 2025Annealing temp should be correct for amplfication
Aman Choudhary
August 24, 2025Annealing temperature was not right
At to high temperature primer fails to bind so no amplification
Payal Gaur
August 24, 2025Annealing temperature not right so primer not bind
Karishma
August 24, 2025Annealing tem.. is not right…so primer not bind
Karishma
August 24, 2025Annealing tem is not right
Dipti Sharma
August 24, 2025Incorrect annealing temperature prevents proper primer binding.
Sneha Kumawat
August 24, 2025Annealing temp. Was not right . High temp hone pr primer bind nhi krenge low temperature hone pr non specific binding hogi
Priyanka Choudhary
August 24, 2025A is correct answer _Annealing temperature was not right because of this low and high temperature pr primer bind nhi hote h and melting point or primer annealing hogi
Sheikh Aleen
August 24, 2025Annealing temperature was not right so primer not bind
Divya rani
August 24, 2025Annealing temperature matters for the DNA amplification
High Annealing temperature results no amplification of DNA or less
Low Annealing temperature results non specific amplification of DNA.
AKANKSHA RAJPUT
August 24, 20251st option is correct
Heena Mahlawat
August 24, 2025Annealing temperature is important for amplification, that’s about 50-70°c
Tanvi Panwar
August 24, 2025Annealing temperature was not right.
Neelam Sharma
August 24, 2025Annealing temperature matters for the DNA amplification
High Annealing temperature results no amplification of DNA or less
Low Annealing temperature results non specific amplification of DNA. And optimize temp for ideal calculation
MOHIT AKHAND
August 24, 2025Done sir ✅
anjani sharma
August 24, 2025Correct annealing temperature is required for the binding of primers
Ayush Dubey
August 24, 2025Annealing temperature was not right.
Sakshi Kanwar
August 24, 2025Annealing temperature was not right , as temperature plays a major role in binding of primer to the specific sequence
Bhawna Choudhary
August 24, 2025Annealing temperature was not right bcz primer binds to the template at optimize Temp
Priyanka Verma
August 24, 2025Annealing temperature should be optimized for binding of primer to the specific sequence
Bharti Yadav
August 24, 2025Annealing temperature was not right.
Falguni Prakash
August 24, 2025Annealing temp
Minal Sethi
August 24, 2025Annealing Temperature was not correct
Priti khandal
August 25, 2025Option a is right annealing temperature is not right
shruti sharma
August 25, 2025Annealing teko was no right
shruti sharma
August 25, 2025Annealing temp was no right
Mitali saini
August 25, 2025The correct answer is (a) Annealing temperature was not right.
Nilofar Khan
August 26, 2025Correct answer is a
Annealing temp was not right
Anjana sharma
August 25, 2025Option 1 is correct annealing required a perfect temperature to bind with primer
Surendra Doodi
August 25, 2025Annealing temperature was not right
Bhavana kankhedia
August 26, 2025Annealing temperature was not right.
Manisha
August 26, 2025Incorrect annealing temperature prevents proper primer binding, leading to no amplification.
Manisha
August 26, 2025Incorrect annealing temperature prevents proper primer binding, leading to no amplification so annealing Temp should be correct for amplification
Devika
August 26, 2025Bcz of incorrect annealing temperature the dba can’t be amplified Best Institute for CSIR NET Life Science Coaching – Let’s Talk Academy”
Rishita
August 26, 2025Annealing temprature was not right
Simran Saini
August 26, 2025Annealing temperature was not right.
Pallavi Ghangas
August 26, 2025No amplification in PCR means an annealing temperature was not right
Mohini
August 27, 2025Option A is correct. Annealing temperature was not correct, that’s why the primers did not bind with the template, and amplification was not done.
Neeraj Sharma
August 28, 2025Annealing tempreture was not right
Kanica Sunwalka
August 28, 2025a nd b – non specific binding agr hue = results in random band not of uniform interval
c -endonuclease – not use in PCR
d- jha pr same seq milega vha pr primer bind kr jaega
Kanica Sunwalka
August 28, 2025ans of 2nd qs
Kanica Sunwalka
August 28, 2025done
Kajal
August 28, 2025Annealing temperature was not right as too high or too low temperature affect the PCR
Muskan singodiya
August 28, 2025Option a
Deepika sheoran
August 28, 2025Option A is correct
Annealing temperature was not Right.
Annealing temperature specific Amplification for target DNA.
Anisha Beniwal
August 28, 2025Option A is correct
Asha Gurzzar
August 30, 2025Annealing temp.was not right,so dna don’t get amplified, primers bind on 50-68°temp
Shobha sharma
August 30, 2025Annealing temparature was not right
Khushi Singh
August 30, 2025Annealing temprature was not ryt
Sonam Saini
August 30, 2025Aneling temperature was not right
Alec
August 30, 2025Annealing temperature was not right.
Yashika Choudhary
August 31, 2025Annealing temp imp
Kirti Agarwal
August 31, 2025Anneling temp is not right
Kajal
August 31, 2025Correct Answer is (a) Annealing temperature was not right
Aartii sharma
August 31, 2025Annealing temprature was not right
Priyanka
September 1, 2025Primers may fail to bind to the template
Konika Naval
September 1, 2025Annealing temperature was not right.
Muskan Yadav
September 5, 2025Annealing temperature was not right.