How to clone cdna with primers having tm difference more than 5 degree C?

Hello,

Your Tms are quite high, and the difference is quite significant.

On my side, when I design my primers I try to have the same Tm for the 2 primers and this in relation to the number of primers and GC (Tm = 2(A+T) + 4 (G +C), I prefer to have a difference in the size of the primers (which will not change anything in the amplification) than to have a difference in the TM (which can, on the other hand, influence the hybridization and therefore the amplification) especially in the case where the difference is quite significant.

Robert Adolf Brinzer

What proportion of your primers are overlapping with your template? You should exclude overhangs from the predicted Tm. Use the lower temp primer for your annealing temp range. Try using a high GC content melting step. Adjust the Mg concentrations. Also have you checked your primers for slef annealing and secondary structure?

Pankaj Yadav

When cloning cDNA using primers with a significant difference in melting temperature (Tm), it is important to optimize the PCR conditions to ensure successful amplification. Here are some steps you can follow to clone cDNA with primers having a Tm difference greater than 5°C:

Designing primers: Design the forward and reverse primers for your cDNA cloning experiment. The Tm of each primer should ideally differ by no more than 5°C to ensure efficient amplification. However, if you have primers with a larger Tm difference, you can still proceed with optimization steps.

Gradient PCR: Perform a gradient PCR to identify the optimal annealing temperature for your primer set. In a gradient PCR, multiple PCR reactions are set up simultaneously, each with a different annealing temperature. The temperature range should cover the Tm values of both primers. By evaluating the PCR products obtained from each reaction, you can determine the temperature at which the primers show the best amplification efficiency.

Touchdown PCR: Alternatively, you can try a touchdown PCR approach. In this method, the annealing temperature is gradually reduced over several cycles. Start with an annealing temperature higher than the Tm of the higher Tm primer and gradually decrease it by 1-2°C per cycle until you reach a temperature suitable for both primers. This allows the primers to bind more specifically during the initial cycles and helps overcome the large Tm difference.

Adjusting primer concentrations: If the Tm difference still poses a challenge, you can try adjusting the concentrations of the forward and reverse primers. A higher concentration of the primer with the lower Tm may enhance its binding and improve the efficiency of amplification. You may need to empirically optimize the primer concentrations through a series of experiments.

PCR additives: Certain PCR additives can help improve amplification efficiency, especially when dealing with challenging primer pairs. For example, adding betaine or dimethyl sulfoxide (DMSO) can reduce secondary structure formation and enhance primer binding. Optimize the concentration of these additives by testing different concentrations in your PCR reactions.

Hot-start PCR: Consider using a hot-start PCR method to reduce non-specific amplification. Hot-start PCR involves adding a heat-activated DNA polymerase to the reaction after an initial denaturation step. This prevents non-specific primer binding during the setup of the PCR reaction and increases the specificity of amplification.

Nested PCR: If the Tm difference between the primers is exceptionally large and optimization methods do not yield satisfactory results, you can consider using a nested PCR approach. In nested PCR, an initial amplification is performed with an outer primer set that has a lower Tm difference. Subsequently, a second round of amplification is carried out using inner primers that specifically bind to the product of the first PCR. This two-step approach can improve specificity and overall amplification efficiency.

Remember to perform appropriate positive and negative controls in your PCR experiments to ensure accurate interpretation of the results. Additionally, consult the literature or seek advice from experienced researchers or molecular biologists who may have encountered similar challenges when working with primers with significant Tm differences.

Daniela Liebsch

Are you trying to amplify directly from cDNA pool and are sure that your gene is expressed well? Maybe a sample with higher expression can help. If it has low expression, and your primers are with overhangs, the "easiest" way is to amplify first with primers that have no overhangs and that 100% fit the template maybe even clone this fragment first, and then in a second step amplify with the primers with overhangs. One thing that can help with large differences in Tm is e.g. do 5-10 PCR cycles with the low annealing temperature (as has been suggested by other replies take the matching part of the primers as a guide only), and then the remaining cycles with a higher temperature corresponding to the entire oligo, possibly even a 2 step PCR with annealing T 72° (you can check Tm calculator webpages online for help which Tm (google your polymerase/company and Tm calculator).

Roshan Shaikh Fakruddin

1. Such a high difference in Tm between the primer pair should be avoided.

2. If there is no option, use the lower Tm of primer to calculate Ta.

3. If the difference in Tm is due to insertion of restriction site (sequences) for cloning purposes, kindly ignore those sequences and re-calculate your Tm and attempt PCR.

4. Check your primer sequences, if you are not able to see any amplification on gel (since you have tried so many versions).

5. What is the product size that you are expecting? Is the size amplifiable by the Taq polymerase that you are using? Check this as well.

6. Check the possibility of RNA degradation. If the RNA is degraded or sub-optimal desired product will not able amplified.

7. Check if the cDNA kit is viable. If the kit is non-viable, PCR won't work.

8. This is remote; check for inhibitors in PCR mix. If amplification is seen with other primers, this can be easily ruled out.

9. If your sequences rich in GC content? If yes, you need to use additives (Formamide, DMSO, betain) to help in resolving it.

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