Table 1.
Test and reference conditions for the optimisation experiments.
| Condition | Preparatory work | Multiplex oligonucleotide ligation | PCR | Hybridisation and analysis on MAGPIX | ||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| DNA isolationa | Probe concentration (nM) |
DNA volume (µL) |
Taq DNA ligase (units) |
Initial denaturation (min) |
Ligation product volume (µL) |
Taq DNA polymerase (units) |
Cycles | PCR product volume (µL) |
Microspheres per reaction |
SAPE concentration (µg/mL) |
Reporter dye | |||||||||||||||||||
| Experiment | 300 | 100 | 50 | IGM | 1 | 2 | 5 | 2 | 4 | 2 | 4 | 6 | 5 | 10 | 3 | 5 | 0.25 | 0.5 | 35 | 40 | 2.5 | 5 | all | 350 | 750 | 2500 | 4 | 10 | SAPE | Alexa 532 |
|
| ||||||||||||||||||||||||||||||
| 1 | R | ns | ls | ls | x | x | x | x | x | x | x | x | x | x | x | |||||||||||||||
| 2 | x | gs | R | gs | x | x | x | x | x | x | x | x | x | x | ||||||||||||||||
| 3 | x | x | R | gs | R | gs | gs | x | x | x | x | x | x | x | x | |||||||||||||||
| 4 | x | x | x | x | R | ls | x | x | x | x | x | x | x | |||||||||||||||||
| 5 | x | x | x | x | x | R | ns | x | x | x | x | x | x | |||||||||||||||||
| 6 | x | x | x | x | x | x | R | gs | x | x | x | x | x | |||||||||||||||||
| 7 | x | x | x | x | x | x | x | R | gs | x | x | x | x | |||||||||||||||||
| 8 | x | x | x | x | x | x | x | x | R | ls | ls | x | x | x | ||||||||||||||||
| 9 | x | x | x | x | x | x | x | x | x | ls | ls | R | ls | R | x | |||||||||||||||
| 10 | x | x | x | x | x | x | x | x | x | x | x | R | gs | |||||||||||||||||
R: reference condition; x: condition used in experiment; ns: test condition which is not significant at α = 0.01 in two-sided hypothesis test; ls: test condition which is significant at α = 0.01 in one-sided hypothesis test with alternative hypothesis true location shift is less than 0; gs: test condition which is significant at α = 0.01 in one-sided hypothesis test with alternative hypothesis true location shift is greater than 0; bold type: optimal condition as concluded from the experiment.
a300: single colony in 300 µL sterile deionised water; 100: single colony in 100 µL sterile deionised water; 50: single colony in 50 µL sterile deionised water; IGM: InstaGene Matrix.