Table 1.
Oligonucleotide sequences for multiplexed P. vivax dbp genotyping for 14 SNPs
| SNP* | Allele* | Tag | Reaction† | Allele-specific oligos‡ | Common oligo‡ |
|---|---|---|---|---|---|
| 308 | G (R) | 73 | 1 | GTAAATAATACAGACACAAATTTTCATAGG (10) | GATATAACATTTCGAAAATTATATTTGAAAAGG (10) |
| T (S) | 46 | GTAAATAATACAGACACAAATTTTCATAGT (10) | |||
| 333 | G (L) | 44 | 4 | CTTTGTTATATCTGTAGTTATTCAACTTAAG (10) | TAATAAATCGCCCTCTACTGC (10) |
| A (F) | 5 | CTTTGTTATATCTGTAGTTATTCAACTTAAA (10) | |||
| 371 | A (K) | 67 | 5 | GAAGGCATCGGATATTCCA (10) | AAGTAGTGGAARATAATTTGCGYAG (10) |
| G (E) | 8 | GAAGGCATCGGATATTCCG (10) | |||
| 375 | T (N) | 88 | 1 | TCCAAAGATGCTRCGCAAATTATT (10) | TTCCACTACTTYGGAATATCCG (10) |
| C (D) | 28 | TCCAAAGATGCTRCGCAAATTATC (10) | |||
| 384 | A (D) | 47 | 2 | CGYAGCATCTTTGGAACTGA (20) | TRAAMAKGCCCAACAGCRTC (160) |
| G (G) | 17 | CGYAGCATCTTTGGAACTGG (20) | |||
| 385 | G (E) | 26 | 3 | GYAGCATCTTTGGAACTGATG (40) | AAMAKGCCCAACAGCRTCG (90) |
| A (K) | 31 | GYAGCATCTTTGGAACTGATA (40) | |||
| 385.1§ | A (K) | 76 | 4 | AGCATCTTTGGAACTGRTRAAA (40) | AKGCCCAACAGCRTCG (40) |
| C (Q) | 80 | AGCATCTTTGGAACTGRTRAAC (40) | |||
| 383.3§ | C (K) | 29 | 5 | TACGAYGCTGTTGGGCC (20) | TKTTYAYCAGTTCCAAAGATGC (90) |
| A (N) | 12 | TACGAYGCTGTTGGGCA (20) | |||
| 390 | C (R) | 45 | 1 | TTCCACCACTGTTTACGAC (10) | GCTGTTGGGCMTKTTYAYCA (10) |
| T (H) | 33 | TTCCACCACTGTTTACGAT (10) | |||
| 417 | A (N) | 16 | 1 | GCAACATTTAATTTACAAATCCATATAAAA (10) | TTCCCCTTTAATCTTTTTTTAACTGAG (10) |
| T (K) | 85 | GCAACATTTAATTTACAAATCCATATAAAT (10) | |||
| 424 | A (L) | 22 | 2 | GGTTCTATATTTACCGCAACATTTAA (10) | TTTACAAATCCATATAAAWTTCCCCTT (10) |
| T (R) | 35 | GGTTCTATATTTACCGCAACATTTAT (10) | |||
| 437 | T (W) | 51 | 3 | GTAAATATAGAACCGCAGATATATAGAT (10) | GGATTCGAGAATGGGGAAGG (10) |
| C (R) | 39 | GTAAATATAGAACCGCAGATATATAGAC (10) | |||
| 447 | G (S) | 59 | 1 | TTKCACTTCTGTGGGCAATTCTG (10) | WYACGTAATCCCTTCCCCATTCTC (10) |
| T (K) | 37 | TTKCACTTCTGTGGGCAATTCTT (10) | |||
| 503 | T (I) | 14 | 2 | GGGATGTTCTGTCAAATAAATTCAT (10) | AAGTGTAAAAAACGCAGAAAAGG (10) |
| A (K) | 68 | GGGATGTTCTGTCAAATAAATTCAA (10) |
All sequences are written 5′ to 3′.
Each SNP uses two allele-specific oligonucleotides that have a unique tag at their 5′ end. The tags are complementary to the anti-tags that are conjugated to the Luminax microspheres (Luminax Corp.). The polymorphic base at the 3′ end of the allele-specific oligonucleotide is underlined and specified under the column labeled allele. Polymorphic nucleotides are shown along with the corresponding amino acid in parentheses. Each common oligonucleotide has a 5′ phosphate and a 3′ biotin. The tag numbers correspond to the 100 fluorescent microspheres that are currently available from Luminax Corp.
Ligation reactions were performed in five separate reactions to limit oligonucleotide interactions since many of the SNPs were only a few base pairs apart.
The concentration of each oligonucleotide for the ligation detection reaction is given in nmol/L within parentheses.
Two mutations are present at codon 386: one at the first position of the codon (386.1) and one at the third position of the codon (386.3).