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

130 Iannone et al.
4. To adjust for well-to-well variability in the wash step, each well may contain a microsphere
population with no cZipCode attached. Subtract the adjusted MFI (as described above)
of this negative control microsphere from the MFI of every microsphere type in that
particular well.
5. Merge the data from the two corresponding alleles and graph the results as x-y coordinates.
4. Notes
1. Oligonucleotide probes for OLA: (1) cZipCodes. We have designed our 58 different
cZipCode sequences to include: (a) 5′ amine group, an 18-atom spacer (CH 3 CH 2 O) 6
to minimize any potential interactions between the oligonucleotide sequence and the
microsphere surface; (b) a common 20-base sequence from luciferase cDNA (5′-CAG
GCC AAG TAA CTT CTT CG-3′) to test for oligonucleotide coupling efficiency; and
(c) a 25-base, non-crossreacting cZipCode sequence derived from the Mycobacterium
tuberculosis genome (7,8) to link each allele of SNP to a particular microsphere population.
The cZipCode sequences have GC-contents between 56 and 72% and predicted Tm values
of 61 to 68°C. Although this chapter does not outline the methodology, biotinylated
cZipCodes may be coupled to Lumavidin-coated microspheres. (2) Reporters. These
oligonucleotides are designed to hybridize to the target sequence immediately downstream
of the capture probe. They are generally 8 to 20 bases in length and contain a 5′ phosphate
group and a 3′ fluorescein modification. The phosphate group is required as a substrate
for the ligase enzyme. Reporter probe Tm range from 36 to 40°C. (3) Captures. The 5′
end of each capture probe contains a 25-nucleotide ZipCode sequence and the 3′ end
contains a 20 to 25 base target-specific sequence that extends to the polymorphic base.
This orientation insures optimal ligase fidelity (12,13). Because each SNP has a minimum
of two polymorphic bases, each SNP will require at least two different OLA capture
probes. If the alleles are assayed in the same reaction volume, these capture probes will
also require different ZipCode sequences. Target-specific capture sequences have a Tm
of 51 to 56°C. (4) Fluorescein-labeled luciferase complement. A 5′ fluoresceinated oligo
complementary to the 20 nucleotides of luciferase sequence in each cZipCode.
2. We have found that only 58 of the 64 red–orange populations Luminex provides could be
run on our FACSCalibur with the Luminex software. The number of useable populations
of microspheres may vary depending upon the cytometer model being used. We have used
all 100 microsphere populations on the LX-100.
3. We have successfully multiplexed over 50 SBCE reactions.
4. Oligonucleotide probes for SBCE: (1) cZipCodes (same as OLA); (2) Captures. Similar to
OLA captures with the exception that target-specific component of SBCE capture probes
is designed to stop just short of the polymorphic base. For SBCE, each SNP will require
only one capture probe. Different alleles are assayed in different reaction volumes. Each
volume will have a different biotin-labeled ddNTP plus the other three unlabeled ddNTPs.
(3) Biotin-labeled luciferase complement. A 5′ biotin-modified oligo complementary to
the 20 nucleotides of luciferase sequence found in each cZipCode.
5. Figure 2 shows representative SNP genotyping results from approx 100 patients for 6 A/G
SNPs analyzed by SBCE on the LX-100. Homozygous and heterozygous clusters are
readily discernible.
Fig. 2. (see facing page) SNP genotyping by SBCE with analysis on the LX-100. SNP
genotyping results from approx 96 patients for 6 A/G SNPs. Biotinylated ddNTPs were used
(one plate for G alleles, one for A alleles) followed by incubation with SA-PE. The results shown
come from an experiment where 35 SNPs were genotyped in two microtiter plates. The points in
the lower left corner represent either negative controls or failed PCR reactions.