Table 3.
Dependence of the amplicon yield on the Tm of the primer in the multiplex PS PCR
| Primer | Tm, °C | Product size, bp | ss loop size, nt | Peak area, arbitrary units |
|---|---|---|---|---|
| no. 2, Table 2 | 60.4 | 200 | 170 | 310 |
| no. 5, Table 2 | 63.4 | 135 | 308 | 1,020 |
| no. 4, Table 1 | 64.7 | 210 | 188 | 2,180 |
| no. 7, Table 2 | 65.7 | 155 | 607 | 2,590 |
| no. 1, Table 1 | 65.7 | 165 | 227 | 4,630 |
The Tm's of the primers were calculated using a nearest-neighbor thermodynamic parameter set (ref. 19, see also http://www.idtdna.com/technotes_facs/Calculating_Tm). Peak areas were determined by analyzing a 14-plex PCR pattern using Fragment manager software provided with the ALF sequencing instrument.