from swill during the washing process. A fluorescence marker is attached to the probe sequence so that the found sequence can be read out by a DNA chip reader.
Figure 1. Sample spot on a DNA chip.
The used primers vary depending on the chip and the experiments. Usually, the primers are between 20 and 100 bases long and they are manufactured synthetically. See [3]for more information.
3 Primer Design
A biological experiment needs a complete primer set that has to be designed. Choosing the optimal primers for a given target sequence or gene requires the evaluation and comparison of several partly independent parameters against a set of ideal values.The user defines the target sequence and specifies a set of ideal parameter values. Each parameter value consists of an ideal value and a range indicating valid parameter results.The minimum and maximum values are used for filtering.They reduce the amount of primers that have to be analysed.A quality score is computed to select the optimum primer.This score is defined as the sum of all distances between the parameter result to the ideal values.
3.1 Hybridization conditions
The parameters taken into account for selecting each optimal primer set have major influence on the quality of the hybridization process where the primers react with the genes or target sequences. The condition is defined by the parameters which are described in the following paragraphs.
Primer length The primer length defines the amount of bases that build the biological primer. Primarily this length
defines the selectivity [6] of the primer. Secondly it has an influence on the melting temperature and the hybridization
effects. The primer length is used to generate the primers from the specified sequence windows that are evaluated. See section 4 for more details.
Melting temperature The primer and the searched gene sequence correspond to each other so that each base can bind to its counterpart. The melting temperature is the temperature at which the bonds between primer and gene dissolves.
This is an important parameter for the PCR and also for the DNA chips to avoid bindings that use only a fraction of the available primer and cause a “false positive” signal.The melting temperature for a given primer p =(p1; :::; pn) is calculated with a prominent approximation for the melting temperature [7, 8, 9, 10]. The formula is:
where R = 1:987(cal=ÆC mol) is the molar gas constant,= 50 ×10-9 is the molar concentration of the primer in its solution, T0 = -237:15℃, and t = -21:6℃ is an empirical temperature correction. The value t may depend upon the ion concentration and other unknown factors.The enthalpy △H(p) and the entropy △S(p) ofthe primer p are computed according to the nearest neighbor schemata[9] and where enthalpy and entropy of a string consist of two bases. The used values for the base combinations are listed in the following Table 1. Thevalues in Table 1 refer to the energy required to disrupt the hydrogen bonds of a single base pair of a paired chain. It is assumed to be influenced by neighboring bases. More details can be seen in [11].
Table 1. Nearest neighbor thermodynamics values [11]
GC content Chemically, hydrogen bonds between the bases of the primer and the gene are responsible for a stable
binding. G-C pairs form three hydrogen bonds and are more stable than A-T pairs which form only two hydrogen bonds. Thus, a high GC content results in a greater stability between primer and gene.
The GC content simply measures the amount of GC bases for the primer. The following formula is used:
Secondary structure Above, only the linear sequence, also known as the primary structure, is considered. Beside
this primary structure also the secondary structure and its effects have to be taken into account. The secondary structure considers the fact that primers are flexible and that base pairs may bind to each other generating structures [6]. The DNA double helix is one example of a secondary structure. Other important secondary structures for the primer design are primer-primer bindings and hairpin, bulge or internal loops. The secondary structure is an important criterion for the selection of the primer, because a hybridization between base pairs can disable the primer for the actual hybridization.
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