Table 1.
Efficiencies of Bio-ASOs Directed to Different Human SNPs
| SNP allele | Typea | Genomic region | Location | Bio-ASO sequence | Competitor ASO sequence | % GCb | Annealing temp.c | Target, kbd | % Yield | Purification |
|---|---|---|---|---|---|---|---|---|---|---|
| BbC | Tv | Minisatellite MS32 | Single Copy | Bio-AATAGGGAGACGATAAAG | AATAGGGAGAGGATAAAG | 39 | 42° | 12.5 | 20 | 200 × |
| BbG | Tv | Minisatellite MS32 | Single copy | Bio-AATAGGGAGAGGATAAAG | AATAGGGAGACGATAAAG | 39 | 42° | 10.2 | 60 | 100 × |
| AB2T | Tv | MHC HLA-DOA region | Single copy | Bio-GTTTGTTGTTTTTTGTTT | GTTTGTTGTTTGTTGTTT | 22 | 35° | 7.0 | 45 | >300 × |
| AB12/5AC | Tv+Ti | MHC HLA-DOA region | In Alu | Bio-AACCCCGTATCCACTGAA | AACCCCGTCTCTACTGAA | 50 | 40° | 7.0 | 50 | >300 × |
| AB13A | Tv | MHC HLA-DOA region | In Alu | Bio-TGAAAACACAAAAAAAAT | TGAAAACACACAAAAAAT | 17 | 35° | 7.0 | 65 | 40 × |
| AB14C | Ti | MHC HLA-DOA region | Single copy | Bio-ACTAATGATTCACTAACA | ACTAATGATTTACTAACA | 28 | 40° | 7.0 | 45 | 300 × |
| JJK6A | Ti | MHC HLA-DMB region | Single copy | Bio-GGTGAGAGGAAAGAAGTG | GGTGAGAGGAGAGAAGTG | 50 | 45° | 6.6 | 65 | 120 × |
| JJK8G | Tv | MHC HLA-DMB region | Single copy | Bio-AACGCGTTGAGCCCACAT | AACGCGTTGACCCCACAT | 56 | 50° | 6.6 | 60 | 120 × |
| JJK10C | Ti | MHC HLA-DMB region | Single copy | Bio-AAAAGGAGTGCGTGTGTG | AAAAGGAGTGTGTGTGTG | 50 | 50° | 6.6 | 60 | 13 × |
| JJK12- | 3 bp indel | MHC HLA-DMB region | Single | Bio-GAAAAAAAGAGGAGTGGA | AAAAAAAGAGAAGGAGTG | 39 | 49° | 6.6 | 60 | 7 × |
| 5053C | Tv | SHOX gene region | Single copy | Bio-CGGCTTTTGCCTTATGGA | CGGCTTTTGCGTTATGGA | 44 | 35° | 9.4 | 30 | 20 × |
| 5543G | Tv | SHOX gene region | Single copy | Bio-GGACGGGCTGGGGTTCCT | GGACGGGCTGCGGTTCCT | 72 | 55° | 9.4 | 30 | >200 × |
Each SNP was tested on restriction enzyme-digested human sperm DNA, using three rounds of DEASH extraction. Yields and purification were assayed as in Figs. 3 and 4. Yields were additionally confirmed for AB2T, JJK6A, and JJK10C by Poisson analysis of single molecule dilutions of enriched DNA (Jeffreys et al. 1998a). Further details of the MHC SNPs can be found in Jeffreys et al. (2001) and at http://www.le.ac.uk/genetics/ajj/HLA/index.html. The SHOX SNPs are described in May et al. (2002) and at http://www.le.ac.uk/ge/ajj/SHOX.
Ti, transition; Tv, transversion; indel, insertion/deletion.
GC content of biotinylated ASO.
Annealing temperatures giving optimum haplotype separation and yield were determined empirically. The relationship between optimal annealing temperature and the GC content of the bio-ASO approximates to 29.6°C + 0.318(%GC)°C.
Size of target released by restriction digestion.