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

Arrays for Genotyping 265
3. Methods
3.1. Array Synthesis (Notes 1–3)
3.1.1. SNP Array Synthesis
1. Clamp a reaction cell (Fig. 2, left, 4 × 40 mm) to a piece of aminated polypropylene using
a G-clamp and a glass or perspex backing plate.
2. Attach ports to the synthesizer and synthesize a patch of oligonucleotide corresponding
to the first allele.
3. Displace the cell by 4 mm so that the cell is alongside the previously synthesized patch.
4. Synthesize the oligonucleotide corresponding to the other allele.
5. Repeat as necessary for other alleles.
6. Place the array in a Duran bottle, add 30% ammonia solution, seal the bottle securely,
and heat at 55°C for 6 h.
7. After allowing the bottle and contents to cool, remove the array, wash with ethanol and
dry. Store at –20°C.
8. The array is cut into strips (1- to 2-mm wide) in a direction perpendicular to the stripes of
oligonucleotides using a scalpel and straight edge.
3.1.2. STR Array Synthesis
1. Clamp a cell (Fig. 2, right, 30 × 40 mm) to a piece of aminated polypropylene using a
G-clamp and a glass backing-plate.
2. Attach ports to the synthesizer and synthesize a patch of oligonucleotide corresponding to the
complement of the flanking sequence before the repeats of the target single stranded DNA.
3. On top of this add a sequence complementary to the smallest number of repeats that may
be present in the target DNA.
4. Displace the cell by 2 mm and synthesize a patch corresponding to one repeat.
5. Repeat step 4 until the maximum required number of repeats has been reached.
6. Place the array in a Duran bottle, add 30% ammonia solution, and heat at 55°C for 6 h.
7. After allowing the bottle and contents to cool, remove the array from bottle, wash with
ethanol, and dry. Store at –20°C.
8. The array is cut into strips (1- to 2-mm wide) in a direction perpendicular to the stripes
of oligonucleotides.
3.2. Single-Stranded Target Production
3.2.1. In Vitro Transcription of RNA
1. Perform PCR under conditions that give the required product.
2. Dilute this product to give a concentration of 0.5 to 1.0 µg/µL.
3. In a mircofuge tube at room temperature add the following reagents in order: 5µ transcription
buffer (4 µL); 100 mM DTT (2 µL); RNasin (20 units); 10 mM ATP, CTP, and GTP
(1 µL each); 250 mM UTP (1 µL); Template DNA (2 µL); [α- 32 P] UTP (2 µL) T7 or SP6
RNA polymerase (20 units); and water to give final volume of 20 µL.
4. Mix and Incubate at 37°C for 1 h.
5. Remove unincorporated label by Sephadex G-25 or G-50 chromatography.
3.2.2. Single-Stranded DNA Preparation
1. Perform PCR under conditions that give the required product.
2. Add T7 Gene 6 exonuclease to give a concentration of 2 U per µL.