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

Microsphere-Based SNP Genotyping 127
2.2. SNP Genotyping by SBCE with Readout
on the LX-100 Flow Cytometer
1. Microspheres (as described in Subheading 2.1.).
2. Template DNA (as described in Subheading 2.1.).
3. Target Probe Primers (as described in Subheading 2.1.).
4. Oligonucleotides (see Table 1 and Notes section): cZipCodes (as described in Subheading
2.1.); captures (as described in Subheading 2.1.); and biotin-labeled luciferase
complement: (Keystone Biosource, Camarillo, CA).
5. Shrimp alkaline phosphatase (2 U/µL) (Amersham Biosciences, Piscataway, NJ).
6. Escherichia coli Exonuclease I (10 U/µL) (Amersham Biosciences, Piscataway, NJ).
7. 2× SBCE reaction mix: (Use 10 µL per reaction) 160 mM Tris-HCl (pH 9.0), 4 mM MgCl 2 ,
50 nM of each capture probe, 2.4 units of AmpliTaq FS (Applied Biosystems, Foster
City, CA), 2 µM of the allele-specific biotin-labeled ddNTP (PerkinElmer Life Sciences,
Inc., Boston, MA), and 2 µM each of the other three unlabeled ddNTPs (Amersham
Biosciences, Piscataway, NJ).
8. 1× SSC containing 0.02% Tween-20.
9. Streptavidin R-phycoerythrin conjugate, 0.1 mg/mL in phosphate-buffered saline, pH 7.2
(SA-PE, Molecular Probes, Eugene, OR).
10. LX-100 flow cytometer (Luminex Corp., Austin, TX), equipped with an XY plate
sampler.
11. Instrument calibration particles for the LX-100 (CL1/CL2 and Reporter Calibrator
Microspheres; Luminex Corp., Austin TX).
3. Methods
3.1. Coupling of cZipCodes to Microspheres (for Microspheres Analyzed
on Either the FACSCalibur or the LX-100)
1. Combine 50 µL of microspheres (2.5 × 10 6 microspheres in 0.1 M MES) with 1 µL of
amino-modified cZipCode oligonucleotide (1 mM in water).
2. At two separate times, add 10 µL of 30 mg/mL EDC in water to the microsphere mixture
(at the beginning of the incubation and then after 30 min).
3. Incubate for 60 min at room temperature with occasional mixing and sonication to keep
the microspheres unclumped and in suspension.
4. Add 200 µL of water containing 0.1% SDS. Vortex and centrifuge 5 min at 1100g.
Carefully remove the supernatant. Add 200 µL of water containing 0.02% Tween 20.
Vortex and centrifuge at 1100g.
5. Remove the supernatant and resuspend the microspheres in 200 µL of TE and store at
4°C (stable for 6 mo).
3.2. Determination of cZipCode Coupling Efficiency (for Microspheres
Analyzed on Either the FACSCalibur or the LX-100)
1. Combine 10,000 coupled microspheres with 3 pmol of fluorescein-labeled luciferase
complement (for microspheres to be run on a conventional cytometer) or biotin-labeled
luciferase complement (for microspheres to be run on the LX-100) in 0.1 mL of 3.3× SSC.
The different microsphere populations may be multiplexed at this point.
2. Heat the microsphere suspension for 2 min at 96°C to denature any secondary structure.
3. Incubate for 30 min at 45°C.
4. Add 200 µL of 1× SSC containing 0.02% Tween-20. Vortex and centrifuge 3 min at
1100g. Carefully remove the supernatant and resuspend in 300 µL of 1× SSC containing
0.02% Tween-20.