Table 4.
Laboratory Working Time and Costs Per Assay for the Four Methods Applied
| BigDye Terminator cycle sequencing kit (Applied Biosystems) | Pyrosequencing PyroMark K-ras (Biotage) | ||
|---|---|---|---|
| Protocol steps* | Time | Protocol steps* | Time |
| 1. Single PCR, including setup | 200 minutes | 1. Single PCR, including setup | 200 minutes |
| 2. Purification and concentration of amplicons | 30 minutes | 2. Single-strand preparation | 30 minutes |
| 3. Sequencing forward and reverse (two reactions) | 20 minutes | 3. Pyrosequencing KRAS codons 12 and 13 | 20 minutes |
| 4. Software analysis | 5 minutes | 4. Software analysis | 5 minutes |
| Total | 4.5 hours | Total | 4.5 hours |
| Detection of mutations | Costs/sample† | Detection of mutations | Costs/sample† |
| Sequence of codons 12 and 13 | ∼$12 | Sequence of codons 12 and 13 | ∼$10.50 |
| Special equipment required | Costs‡ | Special equipment required | Costs‡ |
| ABI sequencer | ∼$100,000 | Vacuum Prep Tool, Pyrosequencer | ∼$150,000 |
| Array LCD-Array K-RASSNP 1.4 (Chipron) | LightMix kit KRAS40–0416 CE-IVD (TIB Molbiol) | ||
|---|---|---|---|
| Protocol steps* | Time | Protocol steps* | Time |
| 1. Single PCR, including setup | 200 minutes | 1. Real-time PCR, including setup | 80 minutes |
| 2. Hybridization of PCR products to LCD arrays | 30 minutes | 2. Analysis of melting curves | 10 minutes |
| 3. 2cd labeling and staining | 10 minutes | ||
| 4. Software analysis | 5 minutes | ||
| Total | 3.5 hours | Total | 1.5 hours |
| Detection of mutations | Costs/sample† | Detection of mutations | Costs/sample† |
| Sequence of codons 12 and 13 | ∼$22.50 | Limited sequence information | ∼$34.50 |
| Special equipment required | Costs‡ | Special equipment required | Costs‡ |
| Array scanner, centrifuge, SlideReader software | ∼$4500 | LightCycler instrument 1.5/2.0 | ∼$30–$75,000 |
*
All protocols start from extracted human DNA. For details see Materials and Methods. Pyrosequencing, Sanger sequencing, and array analysis require standard PCR equipment.
†
Assay costs were calculated excluding Taq polymerase, PCR buffer, and dNTPs. The LightMix kit includes polymerase and controls (total costs ∼$42–$52/patient sample).
‡
Estimated instrument costs as given by the manufacturer or personal communications. For detailed prices manufacturer of local distributors should be contacted.