Why annealing temperature is important in pcr

Learn how to predict and select appropriate melting temperatures for oligo hybridization steps, including PCR. The melting temperature of an oligonucleotide duplex, or T m , is the temperature at which half of the oligonucleotide molecules are single-stranded and half are double-stranded, i. T m is a critical parameter to consider when designing and performing many molecular biology experiments, including PCR and qPCR. Accurate prediction of T m identifies duplexes that are likely to form at specific temperatures, allowing you to determine appropriate thermal cycling parameters.

During the annealing phase of PCR, the reaction temperature needs to be sufficiently low to allow both forward and reverse primers to bind to the template, but not so low as to enable the formation of undesired, non-specific duplexes or intramolecular hairpins, both of which reduce reaction efficiency. Both primers in PCR should be chosen to have a similar T m.

Designing qPCR assays with dual-labeled probes also requires careful coordination of primer T m. When the reaction temperature is lowered from denaturing to annealing during cycling, the probe needs to anneal first to the target. If the probe binds to the target at the same time or after the primers bind, the polymerase may begin replication of target that does not contain bound probe. As a result, new DNA will be synthesized without associated probe degradation and, therefore, will not be detected as an increase in fluorescence.

Such a situation leads to inaccurate data. It is important to check the T m of any oligonucleotide sequences used in PCR even when a previously successful primer and probe set is taken directly from a publication. The design of such published sequences may incorporate T m enhancers such as a minor grove binder or locked nucleic acid bases. These T m enhancers are not necessary for gene expression analysis; unmodified probe and primer sets that provide reliable, accurate data can be designed for the same targets without the added expense of unnecessary modifications.

On the bacterial surface of wild-type E. P-fimbriae and F fimbriae belong to the fimbrial adhesins. Non-fimbrial adhesins are monomeric or trimeric structures that decorate the surface of bacteria. These adhesins are anchored to the surface of the outer membrane and due to their small size, the size of non-fimbrial adhesins is approximately 15 nm, allow an intimate contact between the bacterial cell surface and specific substrates.

One of the major classes of non-fimbrial adhesins is autotransporter adhesins [ 13 ]. P-fimbriae are the most extensively studied adhesins. They are also the first virulence-associated factor found among uropathogenic E. Further receptors for P-fimbriae are present on erythrocytes from pigs, pigeon, fowl, goats and dogs but not on those from cows, guinea pigs or horses [ 19 ].

These fimbriae are encoded in the pap operon, consisting of 11 different genes see Figure 2A : papA bp , papB bp , papC bp , papD bp , papE bp , papF bp , papG bp , papH bp , papI bp , papJ bp and papK bp [ 20 ]. Scheme of pap and F17 operon and annealing sites of the used primers. Genes in the operon are presented as boxes. The positions of used primers to amplify the studied genes are marked with arrows.

A Scheme of pap operon. The scheme of pap operon was drawn based on the GenBank deposited nucleotide sequence X The scheme of F17 operon was drawn based on the GenBank deposited nucleotide sequence L The product of the papA gene is the major subunit protein A In papB a regulatory protein 13 kDa is encoded.

PapB is necessary for the activation of the papA expression [ 21 ]. PapC 80 kDa is located in the outer membrane and forms the assembly platform for fimbrial growth. PapD PapE PapG is the adhesin molecule conferring the binding specificity [ 19 ]. PapH 20 kDa terminates fimbrial assembly and helps anchor the fully grown fimbriae to the cell surface [ 22 ].

PapI 12 kDa is another regulatory protein involved in papA expression due to activation of papB promoter [ 21 ]. PapJ 18 kDa is a periplasmic protein required to maintain the integrity of P-fimbriae [ 23 ]. PapK 20 kDa regulates the length of the tip fibrillum and joins it to the rod [ 24 ]. Many variants of P-fimbriae exist.

PapA molecules from different P-fimbrial serovariants have a high degree of similarity at the N and C termini, while the central portions of PapA exhibit a great variation in the primary structure. This central part of PapA is hydrophilic and exposed and hence under selective pressure from the host immune system.

In addition also P-fimbria-related fimbriae, the so-called Prs-fimbriae, exist. Prs-fimbriae are encoded in the prs pap -related sequence operon [ 18 ]. Ffimbriae are found on pathogenic E. They are mainly detected on bovine and ovine E.

The F17 adhesin binds to N-acetyl- d -glucosamin receptors of bovine intestinal cells; however, F17 subtypes were also found to bind to N-acetyl- d -glucosamin receptors of human uroepithelial and intestinal cells [ 25 ].

F17A protein 20 kDa [ 25 ] is the structural component of the Ffimbriae major subunit protein. F17C protein 90 kDa probably functions as a base protein on which the fimbrial subunits are polymerised.

It functions as the periplasmic transport protein [ 29 ]. F17G protein 36 kDa [ 25 ] is the minor fimbrial component required for the binding of the Ffimbriae to its receptor on the host cell [ 30 ]. Several variants of Ffimbriae exist. The diversity is based on differences in F17A and F17G genes. The variant of Ffimbriae found in humans is designated as G-fimbriae, encoded in the gaf operon [ 25 ]. The analysed 24 clinical haemolytic E. Some more details about the strains are given in Table 1.

As positive control strains, a dog uropathogenic E. Characteristics of the 24 studied E. The erythrocytes are abbreviated as follows: B, bovine erythrocytes; E, equine erythrocytes; C, canine; O, ovine; P, porcine. NT, non-typable. Denaturation time was too long If the denaturation time is too long, DNA might be degraded. Denaturation time was too short If the denaturation time is too short, the DNA will not completely denature and amplification efficiency will be low. For the initial denaturation, use 3 min to activate the polymerase; to denature the template during cycling, use 30 sec.

Back to Top. Having trouble with PCR? Learn more ». Causes Related to Cycling Times and Temperatures Too many cycles were used Excessive cycling increases the opportunity for nonspecific amplification and errors.

Extension time was too long Excessive extension time can allow nonspecific amplification. Annealing time was too long Excessive annealing time may increase spurious priming. Use an annealing time of 30 sec. Annealing temperature was too low If the annealing temperature is too low, primers may bind nonspecifically to the template. Thermal cycler ramping speed is too slow If the ramp speed of the cycler is too slow, spurious annealing may occur due to lower temperature and sufficient time for nonspecific binding.

If ramping speed is not set at the maximum speed for the cycler, increase to maximum ramp rate. Calculated primer T m was inaccurate If the primer concentration is calculated incorrectly, the calculated annealing temperature will also be incorrect. Smeared Bands Back to Top.

Page Contents. Related Content Videos Documents. Number Description Options. Log In to download this document. United States. Using too few PCR cycles can lead to insufficient amplification.

If the extension time is too short, there will be insufficient time for complete replication of the target. If the annealing time is too short, primers do not have enough time to bind to the template. If the annealing temperature is too high, primers are unable to bind to the template. If the denaturation temperature is too low, the DNA will not completely denature and amplification efficiency will be low.

If the denaturation time is too long, DNA might be degraded. If the denaturation time is too short, the DNA will not completely denature and amplification efficiency will be low. GC-rich PCR products are difficult to amplify. To improve amplification, increase the annealing temperature. Template may be sheared or may contain PCR inhibitors. If inhibitors are suspected, dilute existing template; otherwise, use fresh template and increase cycles.

Try a control reaction in which you use a pure plasmid with the addition of the template to determine if any inhibitory effects exist.

Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.

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.