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

Arrays for Genotyping 263
to the array at any position where probe/target duplex contains no mismatches. The use
of enzymes that produce a covalently bonded modification to the array also allows the
assays to be thermally cycled. This increases the signal to noise ratio.
Primer extension/minisequencing/genetic bit analysis has the great advantage that the
reporter (labeled dideoxynucleotide triphosphates) is simple and readily commercially
available. At most four different reporters are needed for each assay. This contrasts
with ligase assays where a unique oligonucleotide reporter is required for each locus
studied. Primer extension can also be adapted for resequencing, using a tiling path of
complementary oligonucleotides.
2. Materials
2.1. Array Fabrication
2.1.1. SNP Array Synthesis
1. DNA synthesizer (Perkin–Elmer/ABI).
2. Synthesis cell (Fig. 2, left).
3. Glass or perspex sheet.
4. Deoxynucleotide 3′-DMT, 5′-phosphoramidites (Glen Research).
5. Reagents for oligonucleotide synthesis (Perkin–Elmer/ABI, Pharmacia, Cruachem).
6. Ammonium hydroxide solution (30%) (BDH).
7. Duran bottle.
8. Aminated polypropylene support (Beckman).
9. Scalpel.
2.1.2. STR Array Synthesis
1. See Subheading 2.1.1.
2. Synthesis cell (Fig. 2, right).
2.2. Target Preparation
2.2.1. In Vitro Transcription of RNA
1. PCR reagents.
2. PCR primer for the target containing a T7 or SP6 promoter sequence.
3. T7 or SP6 RNA polymerase (Promega).
4. Transcription buffer 5µ: 200 mM Tris-HCl, pH 7.9, 30 mM MgCl 2 , 10 mM spermidine,
50 mM NaCl; Promega.
5. DTT (100 mM; Promega).
6. RNase Inhibitor (Recombinant RNasin; Promega).
7. [α- 32 P]UTP (3000 Ci/mmol; Amersham).
8. NTPs: ATP, CTP, and GTP as 10 mM stock solutions, and UTP at 250 µmol (all in nuclease
free distilled water; Pharmacia).
2.2.2. Single-Stranded DNA Preparation
1. PCR reagents.
2. PCR primer for the target strand containing 6 phosphorothioate linkages at the 5′-most
6 phosphate linkages.
3. Thermal cycler (MJ Research).
4. T7 gene 6 exonuclease (Amersham).
5. PCR purification kit (optional, Qiagen).