The PCR or Polymerase Chain Reaction is an in-vitro technique widely used in molecular biology to selectively amplify a piece or a specific stretch of DNA. The target region to amplify is defined by two primers and is called the amplicon. The selectivity of the primers is important to ensure that only the intended region of the DNA template or sample will be amplified and not unwanted parts.

The experimental PCR methodology consists of 20 to 40 cycles during which the temperature of the sample is changed to discrete and selected values corresponding to different biological processes or steps. The steps of a typical cycle are

1. Denaturation step. This is the first step of the cycle. The sample is heated to the melting temperature of DNA, usually around 94-96 °Celcius for 20-30 seconds. The DNA strands separate yielding to single strands for both the DNA sample and the primers.
2. Annealing step. During this step, in which the reaction temperature is lowered to 50-65 °Celcius for 20-40 seconds, the primers and the single-stranded DNA sample bind together.
3. Extension/elongation step. In this step, the temperature is elevated to the optimal temperature of the employed DNA polymerase. The later synthesizes new DNA strand complementary to the DNA sample starting from the 3′ end of the primers and going in the 5′ to 3′ direction. The duration of this step depends on both the used DNA polymerase and on the length of the amplified region.

Additional initial and final steps can be added depending on the exact experimental procedures.

The PCR interface consists of three tabs. One enters the DNA sequence of the sample, called "the reference sequence" in the first tab and then specifies the parameters for solving the PCR design, i.e. to find the desired amplicon in the second tab, the "Parameters tab". Once the prediction is completed, third tab with the results, the "Result" tab, is displayed. The list of available parameters is given below.

• Oligo concentration : concentration of oligonucleotide in the solution, in nM (10^-9M).
• Salt concentration (Na⁺) : concentration of monovalent salt in the solution, in mM (10^-3M).
• Salt concentration (Mg²⁺) : concentration of divalent salt in the solution, in mM (10^-3M).
• Min length : minimal length, in bp, for the primers or the amplicons.
• Max length : maximal length, in bp, for the primers or the amplicons.
• Min T_m : minimal melting temperature, in °C, for the primers or the amplicons.
• Max T_m : maximal melting temperature, in °C, for the primers or the amplicons.
• Min GC : minimal content in G/C bases, in percentage, for the primers.
• Max GC : maximal content in G/C bases, in percentage, for the primers.
• Max GC on 3′ end : maximum occurrence of G/C bases at 3′ end for the primers.
• Max G or C repeated : maximum occurrence of repeated G or C for the primers.
• Max A or T repeated : maximum occurrence of repeated A or T for the primers.
• Max num repeated dinucleotide : maximum occurrence of repeated dinucleotide for the primers.
• Max T_m difference between the primers : Maximal difference between the T_m for the primers defining the amplicon, in °C.
• ΔG method : list of available theoretical method to evaluate the ΔG.
• T_m method for primers/amplicons : list of available theoretical method to evaluate the T_m for the primers and amplicons.
• Max consecutive matches : Maximal number of consecutive matches between a primer and the DNA sequence before considering a binding.
• Max 3′ consecutive matches : Maximal number of consecutive matche between the 3′ end of a primer and the DNA sequence before considering a binding.
• Max percent of matches : Maximal percentage of matches between a primer and the DNA sequence before considering a binding.
• Check using the Amplify method : Check the binding of a primer with the DNA sequence with the Amplify method.
• Min ΔG Hairpin : Discard primers forming a hairpin with an energy below the given ΔG in kCal/mol.
• Min ΔG Hairpin in 3′ : Discard primers forming a 3′ hairpin with an energy below the given ΔG in kCal/mol.
• Min ΔG homoduplex : Discard primers forming a homoduplex with an energy below the given ΔG in kCal/mol.
• Max number of hydrogen bonds : Discard primers forming homoduplex or heteroduplex having a number of hydrogen bonds above the given limit.
• Min ΔG heteroduplex : Discard primers forming heteroduplex with an energy below the given ΔG in kCal/mol.
• Min ΔG cross-dimer in 3′ : Discard primers forming a cross-dimer in 3′ with an energy below the given ΔG in kCal/mol.

The TaqMan procedure is a refinement of the PCR technique as it allows to measure in real time the number of copies of the DNA template present in the PCR. In the classical PCR methodology is possible to measure the evolution of the reaction within the sample tube only at the end of the cycles. With the real-time PCR technique, a single stranded oligonucleotide sequence of 20 to 60 bases complementary to a fragment of the DNA sample belonging to the amplicon, i.e. located between the forward and reverse primers, is added in the sample. This sequence or probe is characterized by the presence of a florescent reporter at the 5' end and a quencher at the 3′ end, both covalently bound. When the probe is bound to the DNA sample, the quencher inhibits the fluorescence of the reporter because of the close proximity of the two. However, during the elongation step of the PCR, whilst DNA is synthesized, the Taq polymerase degrades the probe via the exonuclease and releases the reporter and the quencher, allowing the fluorescence to take place in the tube as the reporter and the quencher separate from each other. The probe takes its name from this process : Taq polymerase + PacMan. The measured florescence in the reaction tube, using LEDs or laser beams, is proportional to the number of released reporters and therefore to the number of copies of the DNA template present in the PCR.

The TaqMan probes interface also consists of three tabs. One enters the DNA sequence of the sample, called "the reference sequence" in the first tab and then specifies the parameters for solving the design, i.e. to find the desired amplicon and probe in the second tab, the "Parameters" tab. Once the prediction is completed, a third tab with the results, the "Result" tab, is displayed. The list of available parameters is given below.

• Oligo concentration : concentration of oligonucleotide in the solution, in nM (10^-9M).
• Salt concentration (Na⁺) : concentration of monovalent salt in the solution, in mM (10^-3M).
• Salt concentration (Mg²⁺) : concentration of divalent salt in the solution, in mM (10^-3M).
• Min length : minimal length, in bp, for the primers, the amplicons, or the probes.
• Max length : maximal length, in bp, for the primers, the amplicons, or the probes.
• Min T_m : minimal melting temperature, in °C, for the primers, the amplicons, or the probes.
• Max T_m : maximal melting temperature, in °C, for the primers, the amplicons, or the probes.
• Min GC : minimal content in G/C bases, in percentage, for the primers or the probes.
• Max GC : maximal content in G/C bases, in percentage, for the primers or the probes.
• Max GC on 3′ end : maximum occurrence of G/C bases at 3′ end for the primers or the probes.
• Max G or C repeated : maximum occurrence of repeated G or C for the primers or the probes.
• Max A or T repeated : maximum occurrence of repeated A or T for the primers or the probes.
• Max num repeated dinucleotide : maximum occurrence of repeated dinucleotide for the primers or the probes.
• More G than C in probe : when checked, the TaqMan probe must have more G bases than C bases.
• G at 3′ end : when checked, the TaqMan probe must have a G base at the 3′ end.
• Max T_m difference between the primers : Maximal difference between the T_m for the primers defining the amplicon, in °C.
• Min T_m difference between primers and probe : Minimal difference between the T_m for the primers defining the amplicon and the T_m for the TaqMan probe, in °C.
• ΔG method : list of available theoretical method to evaluate the ΔG.
• T_m method for primers/amplicons/TaqMan probes : list of available theoretical method to evaluate the T_m for the primers, amplicons, and probes.
• Max consecutive matches : Maximal number of consecutive matches between a primer and the DNA sequence before considering a binding.
• Max 3′ consecutive matches : Maximal number of consecutive matches between the 3′ end of a primer and the DNA sequence before considering a binding.
• Max percent of matches : Maximal percentage of matches between a primer and the DNA sequence before considering a binding.
• Check using the Amplify method : Check the binding of a primer with the DNA sequence with the Amplify method.
• Min ΔG (probe) Hairpin : Discard primers or probes forming a hairpin with an energy below the given ΔG in kCal/mol.
• Min ΔG Hairpin in 3′ : Discard primers forming a 3′ hairpin with an energy below the given ΔG in kCal/mol.
• Min ΔG (probe) homoduplex : Discard primers or probes forming a homoduplex with an energy below the given ΔG in kCal/mol.
• Max number of hydrogen bonds : Discard primers forming homoduplex or heteroduplex having a number of hydrogen bonds above the given limit.
• Min ΔG heteroduplex : Discard primers forming heteroduplex with an energy below the given ΔG in kCal/mol.
• Min ΔG cross-dimer in 3′ : Discard primers forming a cross-dimer in 3′ with an energy below the given ΔG in kCal/mol.

The addition of a minor groove binder (MGB) group to a DNA probe allows the formation of extremely stables duplexes with single-stranded DNA targets. An immediate application is the use of shorter probes in real-time PCR. With respect to their unmodified DNA probes, MGB probes show higher melting temperature and increased specificity, especially when a mismatch occurs in the MGB region of the duplex probe-target. Beside the addition of the MGB group to the DNA probe, the TaqMan MGB procedure is equivalent to the simple TaqMan procedure described before in section IV. a. , on page 35. There are several available MGB groups, however, the software considers currently only the 3' CDPI3 MGB group.

The TaqMan MGB probes interface also consists of three tabs. One enters the DNA sequence of the sample, called "the reference sequence" in the first tab and then specifies the parameters for solving the design, i.e. to find the desired amplicon and probe in the second tab, the "Parameters" tab. Once the prediction is completed, a third tab with the results, the "Result" tab, is displayed. The list of available parameters is given below.

• Oligo concentration : concentration of oligonucleotide in the solution, in nM (10^-9M).
• Salt concentration (Na⁺) : concentration of monovalent salt in the solution, in mM (10^-3M).
• Salt concentration (Mg²⁺) : concentration of divalent salt in the solution, in mM (10^-3M).
• Min length : minimal length, in bp, for the primers, the amplicons, or the probes.
• Max length : maximal length, in bp, for the primers, the amplicons, or the probes.
• Min T_m : minimal melting temperature, in °C, for the primers, the amplicons, or the probes.
• Max T_m : maximal melting temperature, in °C, for the primers, the amplicons, or the probes.
• Min GC : minimal content in G/C bases, in percentage, for the primers or the probes.
• Max GC : maximal content in G/C bases, in percentage, for the primers or the probes.
• Max GC on 3′ end : maximum occurrence of G/C bases at 3′ end for the primers or the probes.
• Max G or C repeated : maximum occurrence of repeated G or C for the primers or the probes.
• Max A or T repeated : maximum occurrence of repeated A or T for the primers or the probes.
• Max num repeated dinucleotide : maximum occurrence of repeated dinucleotide for the primers or the probes.
• More G than C in probe : when checked, the TaqMan probe must have more G bases than C bases.
• G at 3′ end : when checked, the TaqMan probe must have a G base at the 3′ end.
• Max T_m difference between the primers : Maximal difference between the T_m for the primers defining the amplicon, in °C.
• Min T_m difference between primers and probe : Minimal difference between the T_m for the primers defining the amplicon and the T_m for the TaqMan probe, in °C.
• ΔG method : list of available theoretical method to evaluate the ΔG.
• T_m method for primers/amplicons/TaqMan probes : list of available theoretical method to evaluate the T_m for the primers, amplicons, and probes.
• Max consecutive matches : Maximal number of consecutive matches between a primer and the DNA sequence before considering a binding.
• Max 3′ consecutive matches : Maximal number of consecutive matches between the 3′ end of a primer and the DNA sequence before considering a binding.
• Max percent of matches : Maximal percentage of matches between a primer and the DNA sequence before considering a binding.
• Check using the Amplify method : Check the binding of a primer with the DNA sequence with the Amplify method.
• Min ΔG (probe) Hairpin : Discard primers or probes forming a hairpin with an energy below the given ΔG in kCal/mol.
• Min ΔG Hairpin in 3′ : Discard primers forming a 3′ hairpin with an energy below the given ΔG in kCal/mol.
• Min ΔG (probe) homoduplex : Discard primers or probes forming a homoduplex with an energy below the given ΔG in kCal/mol.
• Max number of hydrogen bonds : Discard primers forming homoduplex or heteroduplex having a number of hydrogen bonds above the given limit.
• Min ΔG heteroduplex : Discard primers forming heteroduplex with an energy below the given ΔG in kCal/mol.
• Min ΔG cross-dimer in 3′ : Discard primers forming a cross-dimer in 3′ with an energy below the given ΔG in kCal/mol.

In the Molecular Beacon real-time PCR technique, the probe is an hairpin shaped molecule, or otherwise stated it forms a stem-and-loop structure. The Molecular Beacon probe is designed so that the loop is complementary to the target sequence and the stem allows the opening of the hairpin while hybridizing on the DNA sample, i.e. the hybrid form is more stable than the stem form. A fluorophore and a quencher are covalently linked to the end of the arms or tails. Contrary to the TaqMan probe, the Molecular Beacon probe fluoresces when hybridized and is dark when free in solution. Another important difference with the TaqMan technique is that the Molecular Beacon probes are not consumed during the PCR reaction. Last but not least, Molecular Beacon probes are very specific. This is due to the equilibrium between the hybridized and stem states. A single nucleotide mismatch can be sufficient to favor the stem form of the probe and therefore the probe remains dark.

The Molecular Beacon probes interface also consists of three tabs. One enters the DNA sequence of the sample, called "the reference sequence" in the first tab and then specifies the parameters for solving the design, i.e. to find the desired amplicon and probe in the second tab, the "Parameters" tab. Once the prediction is completed, a third tab with the results, the "Result" tab, is displayed. The list of available parameters is given below.

• Oligo concentration : concentration of oligonucleotide in the solution, in nM (10^-9M).
• Salt concentration (Na⁺) : concentration of monovalent salt in the solution, in mM (10^-3M).
• Salt concentration (Mg²⁺) : concentration of divalent salt in the solution, in mM (10^-3M).
• Min length : minimal length, in bp, for the primers, the amplicons, or the probes.
• Max length : maximal length, in bp, for the primers, the amplicons, or the probes.
• Min T_m : minimal melting temperature, in °C, for the primers, the amplicons, or the probes.
• Max T_m : maximal melting temperature, in °C, for the primers, the amplicons, or the probes.
• Min GC : minimal content in G/C bases, in percentage, for the primers or the probes.
• Max GC : maximal content in G/C bases, in percentage, for the primers or the probes.
• Max GC on 3′ end : maximum occurrence of G/C bases at 3′ end for the primers or the probes.
• Max G or C repeated : maximum occurrence of repeated G or C for the primers or the probes.
• Max A or T repeated : maximum occurrence of repeated A or T for the primers or the probes.
• Max num repeated dinucleotide : maximum occurrence of repeated dinucleotide for the primers or the probes.
• More G than C in probe : when checked, the TaqMan probe must have more G bases than C bases.
• G at 3′ end : when checked, the TaqMan probe must have a G base at the 3′ end.
• Max T_m difference between the primers : Maximal difference between the T_m for the primers defining the amplicon, in °C.
• Min T_m difference between primers and probe : Minimal difference between the T_m for the primers defining the amplicon and the T_m for the TaqMan probe, in °C.
• ΔG method : list of available theoretical method to evaluate the ΔG.
• T_m method for primers/amplicons/TaqMan probes : list of available theoretical method to evaluate the T_m for the primers, amplicons, and probes.
• Max consecutive matches : Maximal number of consecutive matches between a primer and the DNA sequence before considering a binding.
• Max 3′ consecutive matches : Maximal number of consecutive matches between the 3′ end of a primer and the DNA sequence before considering a binding.
• Max percent of matches : Maximal percentage of matches between a primer and the DNA sequence before considering a binding.
• Check using the Amplify method : Check the binding of a primer with the DNA sequence with the Amplify method.
• Min ΔG (probe) Hairpin : Discard primers or probes forming a hairpin with an energy below the given ΔG in kCal/mol.
• Min ΔG Hairpin in 3′ : Discard primers forming a 3′ hairpin with an energy below the given ΔG in kCal/mol.
• Min ΔG (probe) homoduplex : Discard primers or probes forming a homoduplex with an energy below the given ΔG in kCal/mol.
• Max number of hydrogen bonds : Discard primers forming homoduplex or heteroduplex having a number of hydrogen bonds above the given limit.
• Min ΔG heteroduplex : Discard primers forming heteroduplex with an energy below the given ΔG in kCal/mol.
• Min ΔG cross-dimer in 3′ : Discard primers forming a cross-dimer in 3′ with an energy below the given ΔG in kCal/mol.