Hi all, I wish to understand why doesn't all polymerase can detect a single copy of region of interest?
While some polymerase is more sensitive (like dreamtaq), and some are less sensitive, what is the difference if the primer annealing condition is the same? Is it due to affinity? What caused the variation, what is that factor?
How about increasing PCR cycle to detect a single copy? If unspecificity is an issue, then how about utilising touch down PCR first?
-------------------------Answer Update (9/6/2020) -------------------------
To summarise the answers from below, assuming all DNA and water is clean with no contamination in reaction, the lower plasmid number detection limit is different in different companies (like DreamTaq v.s. Q5) due to following reason.
1. The salt composition in different buffer (affecting annealing), possible that manufacturer weight between specificity and sensitivity. We can also see different enzyme recommends using different Tm. Tm-5 to Tm+3.
2. Innate performance of polymerase and response to other additives (accounting to % of actually active taq in solution)
3. The statistical chance to amplify one single plasmid in respond to 1 and 2.
I think that the quality of the pcr primers is more important than the polymerase. Most polymerases are still active at more than 35 cycles which could theoretically generate 35 billion copies but in reality if the primers anneal even slightly in the wrong place the false bands will amplify well and spoil the results. For this reason nested pcr is often used to increase the specificity of the band while maintaining strong amplification. A top quality enzyme with poorly designed primers will still give a bad/wrong result. Touchdown pcr often works well but if the higher annealing primer can anneal in more than one place then it will not help. This can happen with gene families, pseudogenes or even common motifs in the genome.
I think that the quality of the pcr primers is more important than the polymerase. Most polymerases are still active at more than 35 cycles which could theoretically generate 35 billion copies but in reality if the primers anneal even slightly in the wrong place the false bands will amplify well and spoil the results. For this reason nested pcr is often used to increase the specificity of the band while maintaining strong amplification. A top quality enzyme with poorly designed primers will still give a bad/wrong result. Touchdown pcr often works well but if the higher annealing primer can anneal in more than one place then it will not help. This can happen with gene families, pseudogenes or even common motifs in the genome.
As you ask the question, I think it is not a biochemical problem. It is a statistical problem. In every biological sample, there is a dispersion of the analytes with a specific probability distribution. In this case, and according to Poison, below 3 copies per test volume, there is more than a 50% probability that there are 0 copies in a repetition. In theory to detect a single copy, you must carry out 4 independent tests, and at least one will be positive.
It can also to do with the PCR buffer, that comes with the specific Taq DNA polymerase. For example, GC-rich region is generally hard to be amplified. Some PCR buffer is optimized to amplify GC-rich regions, but not for other PCR buffers. So, the region you want to amplify can also be a determinant whether you can get a positive PCR results using a specific Taq DNA polymerase kit. Kenneth Ng
Yuan-Yeu Yau I see, so the buffer composition affects the lowest detection limit? So the polymerase does not really have a strong relationship here? So primers and buffers are the influencing factor. So dream taq engineer the buffer such that it is more sensitive to very low copy, but not the enzyme itself?
Francesc Codony I see, but what explains the variation when comparing different enzyme products? For example dreamtaq is excellent in its detection of very low copy number samples, but some other enzymes are less sensitive. Even if the primer and sample is the same. Does other enzyme sacrifice detection limit for more specific annealing by modification of buffer (instead of the innate property of the enzyme).
Kenneth Ng < Yuan-Yeu Yau, I see, so the buffer composition affects the lowest detection limit? >
What I mentioned was just a possible factor. It can be several factors for that--DNA polymerase itself, the primers you design, the buffer manufacture's produced (ex. Q-solution) or even your template (DNA sample) quality.
I did have similar experience with yours, where the temperate (DNA sample) used was the same, the primers used were also the same. But, polymerase can amplified a specific DNA band, but the other one cannot. After I further purified the DNA samples. Both DNA polymerases could amplify the target. I was wondering why one of the DNA polymeras and its buffer can pick up right sites for amplification in a lower quality DNA sample!?
Senior colleagues already delivered most possible solution, but I think you should also need to check the quality of your DNA first. It is very important! Sometimes, poor quality DNA can lead to the multiple bands after PCR.
Yuan-Yeu Yau I see. If polymerase is a possible factor, I wonder what is the mechanism that affects the detection? Like how come the shape of mechanism of the polymerase affects the lower detection limit?
the PCR, it is necessary to take into account many factors, which affect the final result, among them the quality of the extraction of the ADN o RNA (cDNA) material, the programming of the conditions in the thermocycler which have to be consistent with the primers and the temperatures of efficiency of the polymerase used, as well as the balance of the other components of the master mix, which can affect the amplification of the target fragment. As Paul says, the design of the primers is key, to obtain the amplification. I suggest running a sample that already knows the result so that it can be used as a positive control, which allows you to know if the fault is from the first or the PCR process.
Kenneth Ng < Yuan-Yeu Yau I see. If polymerase is a possible factor, I wonder what is the mechanism that affects the detection? >
That is a good question. I don't know. I have an unpublished data set about this subject. I tried to compare two Taq DNA polymerase (TDP)'s performances--Ex Taq and regular Taq. They were used to amplify a 600 bp fragment. Different conditions were used for amplifications, including (1) purer DNA as temperate, (2) quick isolated DNA (less pure), (3) dilution factors...etc. Ex Taq always gave better results (meaning product was amplified) in different conditions than the other one. Regular Taq could perform as good as Ex Taq when extra step was taken--> i.e. extra step of purification, or dilution.... 'Dilution' can reduce PCR inhibitors and increase amplification efficiency. But, why Ex Taq did not need diluted DNA as template, and were able to amplify a band? Is it resistant to PCR inhibitors? I don't have an answer yet.
It depends on the enzyme purification methods by different companies.
Yuan-Yeu Yau I see. I think different polymerase has different sensitivity to salt and also steric hindrance by inhibitors. And addition to Mohammad Kabir Ahmed 's reply, maybe this could also be a good reason. We always assume that the polymerase from the commercial companies are 100% pure and without anything. So, to conclude from the replies from above, I think the major factors affecting the polymerase would be
1. The salt composition in different buffer (affecting annealing)
2. Innate performance of polymerase and response to other additives (accounting to % of actually active taq in solution)
3. The statistical chance to amplify one single plasmid in respond to 1 and 2.
My additional question to Yuan-Yeu Yau would be, have to tried increasing amount of regular taq to see if the performance improves? And is there an upper limit in the polymerase amount that positively affects the PCR? More taq may simply means more uninhibited taq is present in solution, maybe taq gets more easily inhibited than ex taq. (so to see what is the limiting factor)
Kenneth Ng < Like how come the shape of mechanism of the polymerase affects the lower detection limit? >
The different PCR performance of different kind of DNA polymerase can also due to other components involved. For example, processivity of a DNA polymerase. See below:
Processivity
"In addition to fidelity, DNA polymerases can be evaluated by their processivity, which is the number of nucleotides a polymerase is able to incorporate before dissociating. A polymerase with high processivity binds to the template and extends far and possibly to the end, adding several nucleotides per second. However, most DNA polymerases are intrinsically low in processivity, that they frequently bind to the template and dissociates, adding one nucleotide per second and producing short DNA product per association/dissociation event.
In nature, a processivity factor called DNA clamp is commonly found in organisms to assist the DNA polymerase, to facility timely replication of the large genome. DNA clamps are multimeric proteins in the shape of a ring. It slides along the DNA strand, and its protein-protein interaction with DNA polymerase helps the association between the polymerase and the DNA strand. DNA polymerases with DNA clamps have dramatically higher processivity, capable of polymerizing thousands of nucleotides without dissociating from the DNA template. Scientist also developed DNA polymerases with high processivity by fusing a DNA binding domain to the polymerase, thus maximizing PCR yield and speed, especially for long templates amplification. Processivity is also affected by the buffer conditions such as salt concentration and the sequence of the DNA template ." References can be found in the below link.
https://old.abmgood.com/marketing/knowledge_base/polymerase_chain_reaction_dna_pol_var.php#:~:text=Polymerase%20Chain%20Reaction%20%E2%80%93%20Variations%20of%20DNA%20polymerase,creates%20new%20complementary%20DNA%20strands.
Kenneth Ng < And is there an upper limit in the polymerase amount that positively affects the PCR? >
From Promega. Too much enzyme could cause smear.
" Enzyme Concentration
We recommend using 1–1.25 units of Taq DNA polymerase in a 50μl amplification reaction. In most cases, this is an excess of enzyme, and adding more enzyme will not significantly increase product yield. In fact, increased amounts of enzyme increase the likelihood of generating artifacts associated with the intrinsic 5′→3′ exonuclease activity of Taq DNA polymerase, resulting in smeared bands in an agarose gel (Longley et al. 1990; Bell and DeMarini, 1991). "
Hello!
DNA is one of the components of the chemical reaction of PCR . The higher the concentration of the components in a chemical reaction, the greater its efficiency and vice versa. Thus, the detection of small amounts of DNA is not an easy task. The lower the concentration of DNA in the PCR, the more difficult it is to detect. To overcome the lack of DNA, all other parameters must be optimal.
Theoretically single will be multiplied exponentially. Run PCR in triplicate and analyze statistically.
Alexander Borodin
One can't be fully agreed to your statement. It is not always true that higher concentration = greater efficiency. Higher concentration can also cause inhibition. And also, higher template concentration also means lower efficiency of PCR reaction, because excess templates would act as an limiting factor. There must be an optimal concentration of template in PCR reaction, which is normally in the range 10-30 ng.
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