John M. S. Bartlett.pdf - Bio-Nica.info

128 Iannone et al.
5. For microspheres to be analyzed on the LX-100, incubate an additional 30 min with 5 µL
of SA-PE reagent at room temperature in the dark. Analyze orange fluorescence associated
with the microspheres on the LX-100 without washing. Effective coupling reactions
analyzed on our LX-100 yield 2000 to 4000 mean fluorescent intensity (MFI) units.
6. For analysis of green fluorescence associated with the microsphere populations, analyze on
the FACSCalibur flow cytometer and convert MFI values to molecules equivalent soluble
fluorochrome of fluorescein (MESF) (see Subheading 3.7.). Effective coupling reactions
yield >100,000 MESF after background fluorescence contributed by the microspheres
alone has been subtracted. The corrected MESF value will determine the number of
molecules of cZipCode coupled per microsphere.
3.3. Generation of Target Probes by PCR Amplification
(for Either OLA or SBCE)
1. In a Polyfiltronics 96-well plate, amplify 10 to 20 ng of genomic DNA per well in 15- to
30-µL reaction volumes. Each PCR reaction should contain 1.5 units of AmpliTaq Gold,
400 µM dNTPs, 200 µM forward PCR primer, and 200 µM reverse PCR primer in 1× PCR
buffer I (Applied Biosystems, Foster City, CA). PCR amplifications are uniplexed.
2. Set the thermal cycler to heat 10 min at 95°C min to activate the DNA polymerase,
followed by 40 three-temperature amplification cycles holding at 94, 60, and 72°C for 30 s
each and ending with an additional 5-min extension at 72°C.
3. Hold samples at 4°C following completion of the reaction.
3.4. OLA
1. Incubate the following in a total volume of 10 µL: 1× ligase buffer, 0.1 pmol of each
capture oligonucleotide, 5 pmol of each reporter oligonucleotide, 3 to 20 ng of each
dsDNA target probe (as determined by Picogreen staining, Molecular Probes, Eugene,
OR), and 10 U Taq DNA ligase. Ligation reactions are multiplexed.
2. Incubate in a thermal cycler by heating to 96°C for 2 min, followed by 30 cycles of a
two-step reaction (denaturation at 94°C for 15 s followed by ligation at 37°C for 1 min).
3. Hold samples at 4°C when the cycles are complete.
3.5. Hybridization of Capture Probes to Microspheres after OLA
1. Add cZipCode-coupled microsphere populations (5000 microspheres of each population)
to the ligation reaction (microsphere populations are combined at this step).
2. Adjust the salt concentration to 500 mM NaCl by adding a small volume of 5 M NaCl.
3. Heat the mixture to 96°C for 2 min in a thermal cycler and incubate at 45°C from 2 h
to overnight.
4. Wash microspheres with 200 µL of 1× SSC containing 0.02% Tween-20 by centrifuging
at 1100g for 5 min.
5. Resuspend the microsphere suspensions in 300 µL of 1× SSC containing 0.02% Tween-20
just before flow cytometric analysis.
3.6. Flow Cytometric Analysis of Microspheres Hybridized
to OLA Products on the FACSCalibur
1. Transfer the microsphere suspensions to 12 × 75-mm polystyrene test tubes.
2. Optimize the settings on the flow cytometer to analyze the microspheres using FlowMetrix
Calibration Microspheres in conjunction with Luminex software.
3. Acquire a minimum of 100 microspheres from each population per tube.
4. Analyze a separate tube of calibration particles (Quantum Fluorescence Kit for MESF
units of FITC) using the same instrument settings.