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

Microsphere-Based SNP Genotyping 125
tions of microspheres, manufactured by the Luminex Corporation (Austin, TX), are
identified by their orange and red fluorescent profile. Reporter fluorescence is green.
The second system uses a microsphere-dedicated flow cytometer, also manufactured
by the Luminex Corp., called the LX-100. The two-laser system of the LX-100
uses a red laser (635 nm) to identify the microspheres (red and near infrared emission)
and a green laser (532 nm) to excite the reporter fluorochrome (orange emission).
One hundred individual microsphere populations are available for this system. The
system is available with an XY platform for sampling directly from 96-well microtiter
plates.
2. Materials
2.1. SNP Genotyping by OLA with Readout
on a FACSCalibur Flow Cytometer
1. 2-[N-morpholino] ethenesulfonic acid (MES; Sigma, St. Louis, MO).
2. Microspheres, which are polystyrene beads with a carboxylated surface. Each population
of microsphere has a unique profile of red and orange fluorescence (Luminex Corp.,
Austin, TX). Use 2.5 × 10 6 microspheres in 62 µL of 0.1 M MES (each population in
a separate volume).
3. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Pierce, Rockford,
IL).
4. Water containing 0.1% sodium dodecyl sulphate.
5. Water containing 0.02% Tween-20.
6. Tris [hydroxymethyl] aminomethane hydrochloride (10 mM)/ethylenediamine-tetraacetic
acid (1 mM), pH 8.0 (TE).
7. 3.3× SSC: 0.5 M NaCl, 0.05 M Na Citrate, pH 7.0.
8. Template DNA (human genomic DNA, 10–20 ng, lyophilized).
9. Target probe primers. These should be designed to yield 150- to 500-bp products (see
Table 1).
10. AmpliTaq Gold (Applied Biosystems, Foster City, CA), 5 U/µL.
11. 10× PCR buffer I (Applied Biosystems, Foster City, CA). 10× buffer: 500 mM KCl, 100 mM
Tris-HCl (pH 8.3), 15 mM MgCl 2 , 0.01% gelatin (w/v).
12. dNTPs (Applied Biosystems, Foster City, CA); 10 mM total (2.5 mM of each dNTP).
13. Taq DNA ligase (40 U/µL) and 10× ligase buffer (New England BioLabs Inc., Beverly,
MA). 10× buffer: 200 mM Tris HCl (pH 7.6), 250 mM K acetate, 100 mM Mg acetate,
100 mM dithiothreitol, 10 mM NAD, and 1% Triton X-100.
14. Oligonucleotides (see Table 1 and Notes section): cZipCodes (Oligos etc, Bethel, ME);
reporters (Oligos etc, Bethel, ME, or Biosource/Keystone, Camarillo, CA); captures
(Biosource Keystone, Camarillo, CA); and fluorescein-labeled luciferase complement
(Biosource Keystone, Camarillo, CA).
15. NaCl (5 M).
16. 12 × 75-mm polystyrene test tubes (Becton Dickinson Labware, Franklin Lakes, NJ).
17. FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) equipped with Luminex
Lab MAP hardware and software (Luminex Corp., Austin, TX).
18. FlowMetrix Calibration Microspheres (Luminex Corp., Austin, TX).
19. Quantum Fluorescence Kit for MESF units of FITC calibration particles (Sigma, St.
Louis, MO). Add one drop from each of five separate populations of microspheres (each
population has a different known amount of fluorescein molecules) to 1 mL of phosphatebuffered
saline.
20. QuickCal software (Sigma, St. Louis, MO).