Usually changing Ta for 2 degrees from optimal wouldn't prevent PCR completely, just decrease its efficiency a bit. It's important for quantative PCR, but I guess you need just qualitative, am I right? Are you sure that your template contains the gene for which the fourth primer pair should anneal? Maybe the expression of this gene is usually very low? How many PCR cycles did you use? Did you try to run another PCR with only fourth primer pair and their optimal Ta?

Also, are you sure your primers are good? Did you use Ref-Seq sequence when you designed them? Did you prove that your primers are working using a positive control DNA template? What is your experimental template - genomic DNA or cDNA? Primers designed for cDNA may not work with genomic DNA if they anneal to different exons and there's a very long intron between them.

Also, how did you estimate their annealing temperature? Different calculators use different algorithms to predict primer Ta, and the results may differ ±5ºC. Did you perform temperature gradient PCR with positive control template to adjust primers' Ta empirically? In some cases actual Ta may differ from predicted Ta significantly.

It is also worth checking to see if there are any SNP sites that fall within your primer sequences. This can be particularly problematic if located near the 3' end of your primer and may restrict binding to the DNA template. 

Hi Ekaterina, I am actually using a positive control sample to make sure the primers are working. Also, I am doing another round of PCR with optimal annealing temperature for the said primer pair. I used Primer3Plus to design primers and selected those with mimimum score for primer-dimer and secondary structures. I am using an Optimase polymerase since my aim is to have an amplification without any errors so I used a specially designed protoco for Optimase polymerase. 

Artur, Thanks for your answer. However, the temperatures I mentioned here are all Ta's. As I mentioned in my previous answer, I designed a protocol for Optimase polymerase as recommended by the provider and followed that protocol. 

There are different reasons why a primer pair would not work.

1. Not doing enough to find the right annealing temperature: One can try gradient PCR to find the right annealing temperature.

2. Not designing the primer according to the essential minimum parameters: Please check your primer sequence for GC content (40 to 60%), 3' ends (essentially one or to two G or Cs but nor many) and the Tm of your Forward and reverse primers (they should not differ much, say within 3 deg C).

3. Other reasons could be the genotype you are using may be very different from the reference you used to design the primer; there may be additions; deletions; and SNPs. To resolve this you should have a positive control.

4. Also check what you are amplifying is not a GC rich region; then use specific polymerase that amplifies GC rich region.

Since you are doing many things right, it could be reasons 3 and 4 in your case.

Has anybody amplified this gene from this positive control before? Is it possible that your positive control just doesn't contain the gene (for example, the positive control may be a pool of short DNA fragments and your amplicon may be longer than their average lenght).

If you're completely sure that your positive control sample contains enough gene of interest and you designed the primers carefully, and still you've got no amplification, maybe there is a problem with the sequence that you used as a template while designing primers? If you used "predicted" sequence, it may contain some sequencing errors, so while your primer matches perfectly to predicted sequence there may be actually mismatches that prevent annealing (especially 3'-mismatches). Also, there may be errors in primer sequence if somebody in oligo synthesis lab made a mistake (I myself never experienced that, but still) that also will cause mismatches.

Take a step back and trouble-shoot just ONE pair of primers at a time.  To determine the optimal Tm for a primer pair, you need to run a reaction with an annealing temperature gradient.  Run your products out on a gel and pick the brightest band.

Another issue can be product size.  Larger products need a longer extension time than smaller products.  A good estimate is 30 seconds per 1000 bp of product.  You may need to use a different extension time for each pair of primers.

Do you have a good positive control for both DNA and a set of primers that you KNOW will amplify a product from that DNA?  If yes, include both of those as controls while you trouble-shoot.  Also, you should ALWAYS include a water negative control.

Good luck!

I'd like to share a story about bad positive control template. I designed primers for the first time while I was working in food research institute, and I was given a task to design species-specific primers to discover different domestic animals' DNA in food and reveal food frauds. So, I designed species-specific primers for goat and I purchased some goat meat (in quite respectable shop!) and extracted DNA from it to test these primers, and I got no amplification. I rechecked my primer design, tried to change temperatures, Mg concentrations - and still had no bands on my gels. Finally, I decided to use another positive control and extracted DNA from goat cheese, and there was a good bright band of expected lenght! So it turned out that primers were good but the meat that I tried to use as my first positive control wasn't goat meat at all, so I revealed my first food fraud even before the method design was fulfilled! It's very important to be sure in your positive control samples.

Hi all, I tried doing the PCR with optimum annealing temperature for the primer pairs and it worked well. I am still in the process of optimizing further experiments and will post the results here. Thank you all for your help!