Interaction between primers either for PCR or for DNA chips must be avoided to conserve the maximum sensitivity
of the primer and the spot on the DNA chip.The following four paragraphs show the criteria that are used for the detection of secondary structure effects. The calculation compares the primer sequences and examines the possibility of a hybridization to itself or to another primer. All calculations use the same basic score function for comparing a base pair. This score function is defined as where p = (p1; ::::; pn) and q = (q1; ::::; qm) are the two primers that are analysed for a possible secondary structure.
Self Annealing The self annealing (SA) calculation estimates the possibility of an unintended hybridization to the primer itself. Therefore, the SA score indicates the probability of generating hairpin and internal loops.
The calculation is done using the original primer and an opposite version of the same primer. The following example
shows these two primers.
original primer 5’-TTCGTACGAAC-3’
opposite primer 3’-CAAGCATGCTT-5’
Both primers are defined as
original primer p = {p1; :::; pn}
opposite primer q = {q1; :::; qm}
The calculation of the SA score starts with the left shifted opposite primer, where only one overlapping position with
the original primer exists. It compares each single overlapping position using the score function 3 and accumulates
each single score values to an alignment score. After computing this alignment, the opposite primer is shifted one position to the right and the new alignment score is calculated.This will continue until there is only one overlapping position at the right end of the original primer. The maximum of all alignment scores is used as the SA score for the given primer. The complete function is defined as
The SA score calculation requires the evaluation of all possible alignments. The amount of alignments kSA depend on
the primer length and can be calculated using the formula kSA = (n -1) ×(m-1) 1:
The example in Table 2 shows the calculation of the SA score for the primer p = GATTA. The table shows the
alignments and the resulting SA scores.
Self End Annealing The self end annealing (SEA) calculation is very similar to the SA calculation but it considers
only those alignments where the 3’ end of the original primer belongs to the overlapping region. Furthermore, the
Table 2. Self annealing calculation for the example.
SEA score is accumulated only for these overlaps which are continuous. Therefore, the SEA score evaluates the probability of generating hairpin loops or other primer-primer interaction that start from the 3’ end and are continuous.
Because only these alignments are considered where the 3’ end is involved there are less alignments that have to be
evaluated. The amount is equal to the primer length.The calculation of the SEA score starts in the shift position
where all bases of the original and the opposite primer overlap. All overlapping positions starting from the 3’ end
of the original primer are accumulated using the score function 3. In case that the base pair does not match, the accumulation is aborted. After the alignment score is computed,the opposite primer is shifted to the right by one position and the new alignment score is computed. This process continues until only one position at the 3’ end of the original primer overlaps. The SEA score is the maximum of all alignment scores.
The primer from the SA example is taken to show the computation of the SEA score in Table 3. Altogether,
5 alignments have to be evaluated for the primer p =GATTA to achieve the SEA score. These alignments and the resulting scores are shown.
In case that a primer binds to itself or to a primer with the same sequence a secondary structure is generated. Secondary
structures are also build when several primers are combined for a PCR experiment. Several primers are mixed
Table 3. Self end annealing calculation for the example.
in one tube where each primer can bind to each other and inhibit the reaction with the target sequence or the gene.
Therefore, the effects with different primers must be considered when the optimal primers are selected.
Pair Annealing The pair annealing (PA) calculation takes the interaction of different primers into account and calculates all possible primer pairs. The models and the computing process is similar to the SA tests. Each primer pair is processed using the formula 4.
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