Guidelines for Optimisation of a Multiplex Oligonucleotide Ligation-PCR for Characterisation of Microbial Pathogens in a Microsphere Suspension Array

Table 1

Test and reference conditions for the optimisation experiments.

Condition

Preparatory work

Multiplex oligonucleotide ligation

PCR

Hybridisation and analysis on MAGPIX

DNA isolation^a

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 in two-sided hypothesis test; ls: test condition which is significant at in one-sided hypothesis test with alternative hypothesis true location shift is less than 0; gs: test condition which is significant at 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.