Optimization of the annealing temperature for DNA amplificationin vitro;

Abstract

In the polymerase chain reaction (PCR) technique, DNA is amplified in vitro by a series of polymerization cycles consisting of three temperature-dependent steps: DNA denaturation, primer-template annealing, and DNA synthesis by a thermostable DNA polymerase. The purity and yield of the reaction products depend on several parameters, one of which is the annealing temperature (Ta). At both sub- and super-optimal Ta values, non-specific products may be formed, and the yield of products is reduced. Optimizing the Ta is especially critical when long products are synthesized or when total genomic DNA is the substrate for PCR. In this article we experimentally determine the optimal annealing temperature (TaOPT) values for several primer-template pairs and develop a method for its calculation. The TaOPT is found to be a function of the melting temperatures of the less stable primer-template pair and of the product. The fact that experimental and calculated TaOPT values agree to within 0.7 degree C eliminates the need for determining TaOPT experimentally. Synthesis of DNA fragments shorter than 1 kb is more efficient if a variable Ta is used, such that the Ta is higher in each consecutive cycle.

Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction

“…Only with primer X20161 annealing temperatures too high are calculated (see two bottom rows of Table 5); this is due to a high thermal stability (-71°C) but low specificity of the 5' part of the primer combined with a low thermal stability ( 61°C) but high specificity of the 3' part from which the polymerase has to elongate. Notice that parameters for calculation (14) of Tm, p, are for 100 mM NaCl conditions which seem to be equivalent to the PCR conditions (0.8 mM dNTPs, 1 IG:C = 11.30'C (32) for RNA: IA:U = 20.00°C IG:C = 8.40'C (33) In case of buffers containing Tris, formamide or urea, or of oligonucleotides instead of polynucleotides, correcting formulas are also available from the literature (33 -38). The use of these formulas, especially in combination with each other, can only be seen as a first empirical approximation; therefore an incorporation into the thermodynamics of the program is avoided.…”

“…1C and D the optical melting curve was shifted by 28.8°C in order to correct for the theoretical condition of 1 Between the Tm values of the mobility curves in Fig. 2A and of the temperature gradient gel in Fig.…”

“…denaturation curves of dsRNA as well as of dsDNA are determined experimentally by optical detection or by TGGE and are compared to the appropriate calculated curves. Furthermore experimentally determined optimal annealing temperatures (1) of PCR are compared to calculated denaturation temperatures of primers.…”

Use of reverse transcriptase loop-mediated isothermal amplification assay for field detection of Newcastle disease virus using less invasive samples

“…Collection to use than RT-PCR, since the later requires thermal of cloacal and oro-pharyngeal swabs does not cause profiling and at least 90 minutes to run a reaction farmer resentment, which makes sample collection (Rychlik et al, 1990;Pavlov et al, 2004). Cloacal and easier in different ND outbreaks.…”

PCR Assay for Detection of Staphylococcus aureus in Fresh Lettuce (Lactuca sativa)

“…In this study, the primers features and the correct design lead us to obtain a good and speciic ampliication in a range of 56-60°C. Likewise, a high T a can cause a low or non-ampliication, reducing the possibility to anneal; for this reason, an optimization of priming temperature is necessary [54]. Additionally, annealing was satisfactory at low DNA concentrations (up to 0.5 pg/μl) showing adequate sensitivity.…”