Table 2.
Summary of Long-Range (XL)-PCR Products Generated for Gene Locus Characterization and/or Sequencing
| Fragment ID | Primer sequences | CYP2D6 XL-PCR amplicons | Product size, Kb | Samples∗ |
|---|---|---|---|---|
| 1 (A)† | F: 5′-GTCCCACACCAGGCACCTGTACT-3′ | Nonduplicated gene and gene deletion | 15.6 (nonduplicated) 3.5 (deletion) | HG00337, NA18632, NA18642 |
| R: 5′-GAATTAGTGGTGGTGGGTGTTTG-3′ | ||||
| 2 (B)† | F: 5′-TCACCCCCAGCGGACTTATCA-3′ | 5′ CYP2D6 gene in duplication | 12 | HG00337, NA18642 |
| R: 5′-CCACAGCCCTCAATAAGTGAA-3′ | ||||
| 3 (C)† | F: 5′-CCCTGGGAAGGCCCCATGGAAG-3′ | 3′ CYP2D6 gene in duplication | 12 | HG00337, NA18642 |
| R: 5′-TAGGTAGCCCTGGCCTATAGCTCCCTGACGCC-3′ | ||||
| 4 (E)† | F: 5′-TCACCCCCAGCGGACTTATCA-3′ | CYP2D6–CYP2D7 hybrid gene | 6.7 | HG00337, NA18632, NA18642 |
| R: 5′-TACGGTGGGCTCCCTGCGAG-3′ | ||||
| 5 (D)† | F: 5′-TTGCCACATTATCGCCCGTGAAA-3′ | Full-length CYP2D6 in any arrangement | 8.4 | HG00337, HG04090, HG04206, NA18632, NA18642, NA18973, NA19908, NA20803, NA20875, NA21105 |
| R: 5′-TAGGTAGCCCTGGCCTATAGCTCCCTGACGCC-3′ | ||||
| 6 (A)‡ | F: 5′-TCACCCCCAGCGGACTTATCAACC-3′ | Full-length CYP2D6 in any arrangement | 6.7 | HG00373, HG03225, HG03246, HG03259, HG03780, NA17128, NA18632, NA19777 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)‡ | F: 5′-TGGAGAGAGGCCACCTGAGGTAGTC-3′ | ASXL-PCR of entire CYP2D6 gene (-2609C) | 7.4 | NA19180 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)‡ | F: 5′-CGTCAAGCTTTCCGACATACACG-3′ | ASXL-PCR of entire CYP2D6 gene (-2532G) | 7.3 | NA23878 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)‡ | F: 5′-CCTCCCAAATCTGATGAAAAATATTAATCC-3′ | ASXL-PCR of entire CYP2D6 gene (-2421C) | 7.2 | NA19917 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)§ | F: 5′-GAGGCAACCTGCTCGGG-3′ | ASXL-PCR of entire CYP2D6 gene (-2178G) | 6.8 | NA07439, NA12154, NA17137, NA18642, NA19174 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)§ | F: 5′-CTGTCCTCAGTGGATGATCCCG-3′ | ASXL-PCR of entire CYP2D6 gene (-1770G) | 6.5 | NA23877 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)‡ | F: 5′-CCTGGACAACTTGGAAGAACCG-3′ | ASXL-PCR of entire CYP2D6 gene (-1584G) | 6.4 | HG00337, HG00436, HG00589, HG01086, HG01094, HG02373, HG03781, NA17448, NA18552, NA18973, NA20289, NA21105 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)‡ | F: 5′-CCTGGACAACTTGGAAGAACCC-3′ | ASXL-PCR of entire CYP2D6 gene (-1584C) | 6.4 | HG00337, HG01806, HG03619, HG03781, NA20289 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)§ | F: 5′-CATGGTGAAACCCTATCTCTACTGAAAATAC-3′ | ASXL-PCR of entire CYP2D6 gene (-1426C) | 6.2 | NA17185 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)‡ | F: 5′-TGTGTGTGAGAGAGAATGTGTGCC-3′ | ASXL-PCR of entire CYP2D6 gene (-740C) | 5.5 | NA19239, NA23877 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| 6 (AS-A)§ | F: 5′-TCACCCCCAGCGGACTTATCAACC-3′ | ASXL-PCR of entire CYP2D6 gene (1023C) | 2.9 | NA19239 |
| R: 5′-CCCGAAACCCAGGATCTGGG-3′ | ||||
| 6 (AS-A)§ | F: 5′-TCACCCCCAGCGGACTTATCAACC-3′ | ASXL-PCR of entire CYP2D6 gene (4401C) | 6.3 | NA17185 |
| R: 5′-GACATCTGCTCAGCCTCAACG-3′ | ||||
| 6 (AS-A)§ | F: 5′-TCACCCCCAGCGGACTTATCAACC-3′ | ASXL-PCR of entire CYP2D6 gene (4723G) | 6.6 | NA17176, NA19908 |
| R: 5′-CTGGGAGGTAGGTAGCCCTGACC-3′ | ||||
| 7 (D)¶ | F: 5′-CCAGAAGGCTTTGCAGGCTTCAG-3′ | CYP2D6–CYP2D7 hybrid gene | 8.6 or 10.2‖ | NA18632, NA18642 |
| R: 5′-CGGCAGTGGTCAGCTAATGAC-3′ | ||||
| 8 (B)¶ | F: 5′-CCATGGAAGCCCAGGACTGAGC-3′ | 5′ CYP2D6 intergenic region in duplication | 3.5 | N/A |
| R: 5′-CGGCAGTGGTCAGCTAATGAC-3′ | ||||
| 9¶ | F: 5′-TCACCCCCAGCGGACTTATCAACC-3′ | CYP2D6–CYP2D7 hybrid genes including *36 | 6.1 | NA17287, NA18632 |
| R: 5′-CACCAGAAAGCTGACGACACGAGA-3′ | ||||
| 10 (H)¶ | F: 5′-TCCGACCAGGCCTTTCTACCAC | CYP2D7–CYP2D6 hybrid genes | 5 | NA19785, NA19790 |
| R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′ | ||||
| N/A∗∗ | F: 5′-CCAGAAGGCTTTGCAGGCTTCAG-3′ (1st PCR) F: 5′-GAACCTCTGGAGCAGCCCATACCC-3′ (nested) |
CYP2D6 genes in downstream position | 8.1 (first PCR) 5.0 (nested) | HG00373, NA17222, NA17287 |
| R: 5′-CGGCAGTGGTCAGCTAATGAC-3′ (first PCR) R: 5′-ACTGAGCCCTGGGAGGTAGGTAG-3′ (nested) |
Fragment IDs match those used in Figure 2 to visualize which region(s) each amplicon represents. Forward and reverse primer sequences are as listed (5′ to 3′). A brief description of the region amplified, purpose of the amplification, and fragment lengths also are provided. Samples used to generate respective XL-PCR products are listed in the right-hand column. The position of the single-nucleotide polymorphism that discriminates among alleles is shown in parentheses. The allele-specific nucleotides in primers are underlined.
AS, allele-specific; ASXL-PCR, allele-specific long-range PCR; F, forward; N/A, not applicable; R, reverse.
Samples listed only when sequenced; some XL-PCRs were also run on other samples to determine which allele is duplicated or to confirm structural variation.
PCR as described.25
XL-PCR performed with primers as described.18
XL-PCR performed as described18 with newly designed primer.
This fragment is 8.6 Kb when the duplicated gene has a CYP2D6 (REP6) downstream region and 10.2 Kb if the duplicated gene has a CYP2D7 (REP7) downstream region (see Figure 2).
XL-PCR performed as described.22