Algorithm

In this script, the following rules are applied in the design of the overlapping primers and the primers for the full-length sequence. (1) The two overlapping primers (Fig. 1), each containing the nucleotides from both the two fragments, are partially reverse complemented, with 3′ overhangs (Fig. 2). (2) The Tm values of the four primers should be similar, which facilitates the PCR reactions. (3) The Tm value of the overlapping region between the two primers (Fig. 2) should be moderate. In our program, we set the favorite Tm value 63 °C. However, users can modify this value by changing the variable in the line 90 of the source code (which is ‘Tm_favorite = 63’). (4) The combinations of the four primers are sorted according to the K value, which is defined with the formulae below.

The arrows show the 5′–3′ direction of DNA. The primers are partially complemented with 3′ overhangs. The upper arrow indicates the forward overlapping primer, consisting of the nucleotides (blue) from the 3′ end of the sense strand of the first DNA fragment and the nucleotides (cyan) from the 5′ end of the sense strand of the second DNA fragment. The lower arrow indicates the reverse overlapping primer, consisting of the nucleotides (purple) from the 3′ end of the antisense strand of the second DNA fragment and the nucleotides (orange) from the 5′ end of the antisense strand of the first DNA fragment. The length of each part is labeled, with i, j, k, l all ranging from 0 to 3 bps (3 bps included). The variations of i, j, k, l enable us to walk through all possible combinations for the overlapping primers. The vertical gray dashes show the overlapping region between the two primers, which is also used for bridging during overlap extension PCR to acquire the full-length chimera DNA sequence.

The ‘Tm’ represents the value of melting temperature of primer. The ‘primer5’, ‘overlap3’ are the primers for the first DNA fragment to be joined and ‘overlap5’, ‘primer3’ are the primers for the second DNA fragment to be joined. The ‘overlap’ is the overlapping region between the two primers. The ‘Tm_favorite’ is the set optimal Tm value of the overlapping region between the two primers (Fig. 2). A lower K value of the combination indicates that the difference in melting temperature among the four primers is smaller.