I have been trying to amplify one of the gene by PCR and I have optimised the conditions for the same and have got an amplicon. To confirm if the amplicon is my desired gene, I am using PCR product as my template and doing PCR. But I am unable to get the amplicon when I use PCR product as template. Every time I use Genomic DNA as template it works but with all same conditions PCR product as template doesn't work. What might be the reason for this?
your problem is probably over amplification which produces a smear of longer pcr products because you have too much template. 20 cycles of pcr is about 1000000 times more product and at this level product molecules self anneal and produce smears instead of a band. Try amplifuing a dilution of your first pcr product of 1/100 or 1/1000. Amplify 1ul of diluted dna and only run about 20 cucles. Ideally set up 6 identical re pcrs and remove a tube at 16, 18, 20 etc cycles to see what gives a good clean amplification
Hello,
To confirm that your amplification product is indeed your target gene sequence, I advise you to carry out sequencing instead, to do a 2nd verification by PCR?
Additionally, even if your sequence was amplified by PCR, it is not certain that your sequence is 100% the same due to replication errors that can be caused by Taq polymerase.
Nested PCR is where the template is a PCR product itself. However the second set of primers should be designed internal to the first set. In order to confirm the PCR product, sequencing is the only way out.
The problem is in the primer-dimer that is always formed during the first PCR round using genomic DNA. The longer the PCR amplicon, the more serious the problem of the second round of PCR using the PCR amplicon from the first round. Sudha Anandt is absolutely correct about applying nested PCR in the case when a PCR product is used as a starting template. However, Sudha does not explain the reason for the 'second' PCR failure. The efficiency or yield of the primer-dimer amplification is almost quantitative (nearly 100%), always greater than the efficiency of the target amplicon, and because of that primer-dimer, formed in the first round of PCR, overruns the target amplicon. In my recent study, I found that the efficiency of primer-dimer amplification can be even greater than 100% (more than double per cycle). I found the mechanism, but it is a long story to tell here. Just trust me and always follow the rule: if a PCR amplicon is your starting template - always use nested PCR for the second round of PCR.
If you are successfully obtaining an amplicon using genomic DNA as a template but not with PCR product as a template, several factors might be causing this issue:
1. Template Quality and Purity:
- The PCR product might contain impurities that inhibit the second PCR. These could include remnants of dNTPs, primers, enzymes, or buffer components from the first PCR reaction.
- Ensure that the PCR product is adequately purified before using it as a template in subsequent PCR reactions.
2. Template Concentration:
- The concentration of the PCR product may be too low or too high. Too much template can lead to inhibitory effects, and too little may fall below the detection limit.
- Quantify and dilute your PCR product to an optimal concentration for use as a template.
3. Primer-Dimer Formation:
- The initial PCR could have produced primer-dimer artifacts that are preferentially amplified in subsequent PCRs, especially if the primer concentrations are not optimized.
- Use a hot-start PCR enzyme and optimize primer concentrations to minimize primer-dimer formation.
4. Secondary Structures:
- The PCR product may form secondary structures that inhibit primer binding or extension.
- Consider using a PCR additive like betaine or DMSO to help resolve secondary structures.
5. Primer Specificity:
- The primers may not be annealing specifically to the desired regions on the PCR product.
- Check your primer design and consider redesigning the primers if necessary, ensuring they are specific to the regions flanking your gene of interest.
6. Enzyme Exhaustion:
- The DNA polymerase from the first reaction might be carried over and become exhausted or inhibited in the second PCR.
- Ensure that you're adding fresh polymerase for the second PCR.
7. Change in Reaction Conditions:
- Even if all conditions seem to be the same, slight variations in magnesium concentration, buffer composition, or pH levels when using PCR product as a template might affect the outcome.
- Re-optimize the PCR conditions for the PCR product as a template.
8. Allelic Dropout or Preferential Amplification:
- In some cases, especially when dealing with highly similar sequences, one allele or sequence variant may be preferentially amplified over another.
- If relevant, check for the presence of sequence variants that might affect primer binding.
To troubleshoot this, consider the following steps:
- Purify the PCR product before using it as a template.
- Perform a dilution series of the PCR product to find an optimal concentration.
- Ensure that the primers used in the second PCR are not complementary to any regions within the amplified product that could lead to primer-dimer formation or secondary amplifications.
- You might also consider a nested PCR approach, where the second set of primers anneals within the first PCR product, reducing the chances of non-specific amplification.
Finally, to confirm that the amplicon is indeed the gene of interest, you could also consider sending the initial PCR product for sequencing rather than performing a second PCR. This would give you a definitive answer to the identity of your amplicon.
Deepak Thippest has outlined a comprehensive set of troubleshooting steps to address common issues encountered during PCR procedures. It's essential to clarify that the original query, posed by Sahana G Ravikumar, revolved around discriminating the disparities in amplification between genomic DNA and an amplicon derived from genomic DNA in the initial round of PCR. In the context of clarifying this distinction, the discussion narrows down to PCR amplification between these two specific scenarios, namely, starting from genomic DNA versus utilizing the amplicon derived from genomic DNA as a seed template to enlarge the quantity of the same amplicon.
Among the eight items or potential issues listed in the troubleshooting guide, it is worth excluding seven of them, such as primer design, the preparation of PCR mixture components, optimization of PCR time/temperature profiles, and more. These elements can be disregarded because we are primarily addressing the same PCR process while altering only the initial material. As mentioned earlier, this leaves us with the most prevalent cause of PCR failure, which is primer-dimer formation. It is crucial to understand that primer-dimer formation occurs in virtually every PCR reaction, whether it is visible/detectable or not.
When employing the amplicon from the initial round as the starting material, it is important to note that this amplicon is already contaminated with primer-dimer formed during the first original PCR round. To achieve satisfactory yields during the second round of PCR, the recommended approach is to utilize a nested set of primers, as advised by Sudha Anandt (discussed previously). This strategic adjustment is crucial for successful amplification when working with the amplicon obtained from the initial PCR round.
Please confirm your first PCR on agarose gel and also quantify it then go for second PCR. May be the PCR product having concentrated template.
Sahana G Ravikumar Could probably be due to inadequate template DNA concentration, low primer quality, improper primer annealing temperature, or polymerase inactivation. Kindly check and optimize these factors. Many thanks. Sahana G Ravikumar
Insufficient amplification can result if the initial amount of template is too low. Increase the number of amplification cycles in increments of 5, or, if possible, increase the amount of template. Contaminants in the dNTP mix can lead to incomplete or incorrect amplification or PCR inhibition. Use high-quality dNTPs.
The PCR reaction product is a very concentrated suspension of the amplified fragment at the end of successful amplification, so one needs to use a very diluted aliquot of PCR reaction as the template.
I think you should elute oùt and purify the PCR product by column method.
you should calculat the concentration of the tamplet DNA using the following question; C1*V1=C2*V2.
in which C2 is the concentration that you should use in the final PCR volume
1. Try diluting the sample after checking he concentration in nanodrop. (Your PCR product is usually concentrated)
If that doesn't work
2. Check the purity of the sample after you elute out the product. Sometimes the elution salts interfere with the PCR process.
If there is insufficient template at the beginning, there may not be enough amplification. Either increase the amount of template, if possible, or increase the number of amplification cycles in steps of five.
It sounds like you are using to much template and overamplifying. Keep in mind your PCR product concentration is much higher than your genomic template. I would recommend checking your DNA concentration by nanodrop and dilute to a concentration similar to your genomic DNA concentration and then you should be fine.
Your problem is amplification (20 cycles of PCR increases the product by about 1,000,000 times).
Therefore, as was explained by one of the esteemed researchers, you must try to dilute the first PCR product by 1/100 or 1/1000. Amplify 1ul of diluted DNA.
There is a well-known rule: never use PCR products as a seeding point in all consequent PCR reactions unless you use another "nested" set of primers. That is because, in most cases, primer-dimer generated and well amplified in the first round of PCR, overrun target amplification in the second round of PCR.
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