If the results of electrophoresis show clear bands for the amplified VL and VH fragments, it suggests that the PCR amplification step is working correctly, and the templates are being successfully amplified. And If you have already contacted the oligo provider and they have confirmed that the electrospray analysis of the primers looks clean, it suggests that there may not be an issue with the primer sequences themselves. I think it would be effective if you check these consideration as well:

Primer concentration: Take care to use an appropriate primer concentration in the PCR reaction. Insufficient primer concentration may lead to incomplete amplification and the occurrence of missing bases in the primer regions. Conversely, excessive primer concentration can result in nonspecific amplification and other complications.

PCR conditions: Evaluate and optimize the PCR conditions, which include the annealing temperature, extension time, and cycle number. Suboptimal PCR conditions can contribute to incomplete amplification or errors in the amplification process.

DNA polymerase fidelity: Consider experimenting with alternative DNA polymerases that possess higher fidelity. Although GoTaq polymerase is commonly used and generally reliable, using a different polymerase may help alleviate the issue of missing bases in the primer regions.

Contamination: Thoroughly check that all reagents and equipment used in the PCR reactions are free from contamination.

Sequence analysis: Perform a detailed analysis of the sequencing data. Pay particular attention to any patterns or specific regions where missing bases are observed. This analysis may facilitate the identification of potential sequence motifs or secondary structures that might impact primer binding or extension.

Dear Aliakbar,

thank you for your hints.

We tested several polymerases so far and GoTaq gave us the best results in terms of yield and product purity.

We however did not analyze the sequences of the products.

As for the conditions we are using the standard conditions from the manual.

What in my eyes brings me back to synthesis errors is that we have one primer from a different batch. This primer never prudces sequences with a missing base in the overhang or primer binding region.

The primers may not be designed correctly or PCR conditions may not be optimized. cDNA pool must be good quality.

Use high quality primers, fresh reagents, and perform a negative control reaction to confirm that the problem is not due to contamination.

Perform a DNA sequencing reaction on the PCR to determine the exact sequence, and to identify any errors that are present.

The problem is most likely due to synthesis errors in the other primers.

Dear Normann,

You are right, so by noting that this primer consistently produces sequences with missing bases in the primer binding region, it suggests that there may be some specific issue related to this particular primer batch that is causing the problem. Further investigation, such as sequence analysis and primer quality assessment, can help determine if the primer from the different batch is indeed responsible for the observed missing bases and guide appropriate actions to address the issue.

The randomly missing bases in the primer binding region or overhang after amplification of VL and VH fragments from a cDNA pool could be due to several factors. One possibility is the presence of template secondary structures that are causing the polymerase to pause or terminate extension. This can occur if there are regions of high GC content or secondary structures such as hairpins or stem loops in the template. Another possibility is errors during the PCR amplification step, which can introduce mutations or deletions in the product.

Other factors that could contribute to the issue include suboptimal PCR conditions such as incorrect annealing temperatures or extension times, improper template quantification, or the presence of inhibitors in the reaction. It may be necessary to optimize the PCR conditions, including the annealing temperature, extension time, and MgCl2 concentration, to improve the specificity and yield of the desired product.

It is also possible that the issue could be related to the purification or cloning steps. Contaminants in the DNA preparation, such as RNases or genomic DNA, could interfere with the PCR reaction, while errors during the cloning step could result in the insertion of incorrect sequences.

To troubleshoot the issue, it may be helpful to analyze the PCR products by gel electrophoresis or sequencing to confirm the presence of missing bases. Additionally, repeating the PCR amplification using a different polymerase or adjusting the PCR conditions may help to identify the root cause of the issue.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

Dear Nikolay.

Thank you for your suggestions.

Yet the errors mostly occur in regions connected to one of the primers (binding site or overhang) So template sec. structures most likely will not be the cause. Also I my eyes stemloops would cause deletions of whole patches, byproducts as well as inferior yield which is not the case (checked several times by gel)

It is known that the GoTaq produces indels at some frequency (~10E-6), which alone cannot explain why almost all sequences have one or more indels (mostly in the primer region)

What we plan to do ist ordering primers (a subset for controls) from another distributor and reamplifying the VHs and VLs using some further polymerases and directly cloning them into pJet for sequencing (so no overlap extension PCR etc this time). If there is a problem with the primers we will see it, if there is a problem with the polymerase we will see it too.

The nature of the error (single base indels), its distribution across the product (mostly primer regions), its frequency (>80-90% products with one or more errors) as well as the randomness of the error could not yet be explained by either the primer manufacturer or the polymerase manufacturer.