Table 1. cDNA–PCR for full-length pyrosequencing with overlapping amplicons.
| Amplicon | Primer Name | Gene-Specific Sequencea | Product Size (bp)b |
|---|---|---|---|
| 5′ UTR | 5′ UTR | 5′-AGAGTCTCCTCAGACGCCGAG | 546 |
| 5′ Leader | 5′ Leader | 5′-CCCCGAACCCTCCTCCTG | 509 |
| 5′ R | 5′-CGTTCAGGGCGATGTAATCC | ||
| Internal | Int F | 5′-AGGGKCCGGAGTATTGGGA | 547 |
| Int R | 5′-TGATCTCCGCAGGGTAGAAGC | ||
| 3′ UTR | 3′ F | 5′-ACCCAGCGSAAGTGGGA | 698-702 |
| 3′UTRa | 5′-CCTCGCAGTCCCACACAAG | ||
| 3′ UTRb | 5′-CCTGCTTCTCAGTTCCACACAAG | ||
| 3′ UTRc | 5′-CTGCATCTCAGTCCCACACAAG | ||
Primer sequences were adjusted to obtain an average Tm of 63°C so that all PCR reactions could be performed under a uniform set of thermal cycling conditions as described previously (Wiseman et al. 2009).
a
In addition to the gene-specific sequences shown here, the 5′ end of each forward and reverse primer contains a GS FLX Titanium (Lib-A) adaptor A or B, respectively as well as a 10 bp Multiplex Identifier (MID) sequence
b
Amplicon lengths include 70 bp for GS FLX Titanium (Lib-A) adaptors and MID's