Table 1 Comparison of screening BAC library by conventional PCR and by MT-PCR-HRM.
Compared features | Screening by conventional PCR using 3D pooling strategy | Screening by MT-PCR-HRM using 2D pooling strategy |
|---|---|---|
Maximum number of 384-well plates to be pooled in one super pool | 10 384-well plates | 25 384-well plates |
Number of super pools to be tested to identify a positive superpool (N: The total plate number of the BAC library) | N/10 | N/25 |
PCR reactions (PCR/PCR-HRM) needed to identify a positive super pool | N/10 | N/25 |
Reactions needed to identify the plate ID for one positive super pool | 10 | 10 |
Reactions needed to identify the clone ID from one positive plate | 40 | 18 |
Total number of reactions needed to get positive BAC clone ID from whole library | N/10 + 50 | N/25 + 28 |
Multiplexing possibility | No | Yes |
Checking on agarose gel | Needed | NO |
Cost | 0.16 £/1 PCR reaction + cost for agarose gel electrophoresis | 0.17 £/1 PCR-HRM reaction |
Procedure duration to anchor 1 marker | 12 h | 3 h |
Scoring data | Manually from gel photos | Semi-Automatic (figures and summary tables can be exported from HRM rotor system) |