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

Microsphere-Based SNP Genotyping 129
3.7. Analysis of FACSCalibur Data
1. Convert all green fluorescence measurements from MFI to MESF by either manually
creating a calibration curve from the fluorescent intensities of the calibration particles
or by using QuickCal software.
2. Adjust raw MESF values by subtracting the microsphere alone control MESF values
to eliminate microsphere-contributed background fluorescence. This is necessary, because
each microsphere population in the 64-microsphere set emits a small amount of fluorescence
in the green reporter channel. Microsphere corrections range from 1000 to
30,000 MESF, depending on the microsphere population.
3. To adjust for tube-to-tube variability in the wash step, one may include a microsphere
population with no cZipCode attached. Subtract the adjusted MESF (as described previously)
of this negative control microsphere from the MESF of every microsphere type in
that particular tube to normalize the data.
4. Merge the data from the two corresponding alleles and graph the results as x-y coordinates.
3.8. SBCE
1. PCR amplification of target probes is performed as described in Subheading 3.3.
2. To degrade the excess PCR primers and dNTPs before the SBCE assay, add 1 unit of
shrimp alkaline phosphatase and 2 units of E. coli Exonuclease I directly to 10 µL
of pooled PCR products (10 to 20 ng of each amplicon) and mix thoroughly.
3. Incubate at 37°C for 30 min and then for 15 min at 80°C to inactivate the enzymes.
4. For each SNP, set up similar reactions differing only by the choice of labeled ddNTP.
Add 10 µL of pooled, treated PCR products to 10 µL of SBCE reaction mix (160 mM
Tris-HCl, pH 9.0; 4 mM MgCl 2 ; 50 nM of each capture probe; 2.4 units of AmpliTaq
FS; 2 µM of the allele-specific biotin-labeled ddNTP; and 2 µM each of the other three
unlabeled ddNTPs).
5. Denature the reactions at 96°C for 2 min and follow with 30 amplification cycles at 94°C
for 30 s, 55°C for 30 s, and 72°C for 30 s.
6. Hold reactions at 4°C.
3.9. Hybridization of Capture Probes to Microspheres after SBCE
1. To the wells of a standard 96-well microtiter plate in a total volume of 30 µL, add 1000
of each cZipCode-coupled microsphere population to 20 µL of SBCE reaction mixture.
Adjust salt concentration to 500 mM NaCl and 13 mM EDTA.
2. Incubate the mixture at 40°C for 1 h.
3. Wash the microspheres with 150 µL of 1× SSC containing 0.02% Tween 20.
4. Centrifuge for 5 min at 1100g and remove the supernatants.
5. Resuspend microspheres in 60 µL of 1× SSC containing 0.02% Tween 20.
6. Add 5 µL of SA-PE to the microsphere-hybridized SBCE reaction products.
7. Incubate the mixture for 30 min at room temperature.
3.10. Flow Cytometric Analysis on the LX-100 and Data Analysis
1. Optimize the settings on the LX-100 to analyze the microspheres using Luminex calibration
particles in conjunction with Luminex software.
2. For each microtiter well, analyze a minimum of 30 microspheres of each population.
3. Adjust the raw MFI values for microsphere background fluorescence by subtracting microsphere
alone control MFI values from the MFI value of each corresponding microsphere sample.
This is necessary because each microsphere population in the 100-microsphere set emits
a small amount of fluorescence into the orange reporter channel. Microsphere corrections
range from 1 to 100 MFI, depending upon the microsphere population.