TABLE 1.
PCR primer sequences
| Oligonucleotidea | Sequence |
|---|---|
| DU1 | GGCTACCGNCWSMTCGACAC |
| DL1 | GGGTGSAGCTCGAYCTGGTT |
| 14nU1 | CTATGACAATGAGGCAGAGGTC |
| 14nU2 | CGCGCGCGAGGAAGTTTTTGTGACA |
| 14nU3 | CCGTGCCCGAAGCAAGACAA |
| 14nL1 | GCTGCAAGAGCTTCTCGAGATC |
| 14nL2 | AGTTCGAGACTCCGATGCCCTTAAC |
| 14nL3 | CGAATGCGTGCCAAGTCTCAA |
| 14nL4 | GACCTCTGCCTCATTGTCATAG |
| 28nU1 | TTATGACAACGAGGCCGAGGTT |
| 28nU2 | GCCATTCAAGAGTCGGTCGACA |
| 28nU3 | CGAAACCGGATTGGTGAAATCA |
| 28nL1 | ACAACATTCGCAGCCGCAAGAA |
| 28nL2 | GAAAGTTTGAGACACCGATTGAT |
| 28nL3 | ACCGATTGATTTCACCAATCCG |
| 28nL4 | TTGCCTCGTGGTATCCGTGGCG |
| 14fU | GCCGTTTTCGCTGTCACCTA |
| 14fL | TTTCTTCGTCCAGGGGAGTTTG |
| 28fU | TCGGCCCGTGGAGCCAAAAC |
| 28fL | TCGCGCTCTGAATCGTTCTG |
| 14expU | GAGAACAATTGTATGAGCGCAGAACAGCCT |
| 14expL | TCTTCTAAGCTTCACTAATTCATATCGTCAGGATT |
| 28expU | GAGAACAATTGTATGGCATCGCCGCTGGTT |
| 28expL | TCTTCTAAGCTTCACTAATTCATGTCGTCTGGGTT |
a
Primers designated D were degenerate primers used to generate internal fragments of the genes. U or L refers to the upper or lower DNA strand. Those with the letter “n” are the nested primers used in generating the flanking regions of fragments 14 and 28. Nested primers ending with the number 1, 2, 3, or 4 were used in the first, second, third, or fourth round, respectively. The set with the letter “f” amplified the full-length genes from environmental DNA, and that with the letters “exp” introduced restriction sites to allow introduction of the genes into an expression vector (Materials and Methods). The locations of the primers in the assembled gene and flanking regions are illustrated in Fig. 2.