Table 2. Primer sequences and PCR conditions for SPARC sequencing.
| |
Primer sequence |
|
|
|
|
|---|---|---|---|---|---|
| Amplifying target | Forward primer (5′→3′) | Reverse primer (5′→3′) | MgCl2 (mM) | Ta (°C) | Size (bp) |
| SPARC-1 (promoter + exon 1) |
CCAGTTCCAAATCATCAAGGA |
GGGGTTGGTGCAACTATAGAA |
1.5 |
59 |
668 |
| SPARC-2 (exon 2) |
AAATGGAACCAACCTCCTCA |
CAATGGTCCTCATCCCAGTT |
1.5 |
60 |
388 |
| SPARC-3 (exon 3) |
AGCTCCCCTAGCCTGTATCC |
CCCTAATTTCTCAGGGCACA |
1.5 |
60 |
225 |
| SPARC-4 (exon 4) |
CTTTCCCTAACACCCCTGGT |
TCATGTAGGCTGTCCTCGTG |
1.5 |
60 |
367 |
| SPARC-5 (exon 5) |
TGTGCTAGTCCAGGTGATGC |
TGTATTCCGAAGTGCCCAAT |
1.5 |
60 |
222 |
| SPARC-6 (exon 6) |
CAGTGTCCCCATCTCTGAAA |
CCCAAGACAGGAGTCTGGAA |
1.5 |
60 |
250 |
| SPARC-7 (exon 7) |
AAGAAACTGTGGCCTGGAGA |
CTGGTGCTCAGGGGTAAATG |
1.5 |
60 |
396 |
| SPARC-8 (exon 8) |
CTGGCTAGTCTCTGCCTGCT |
TCACTCTAGGGTCTGGGGTCT |
2.0 |
60 |
279 |
| SPARC-9 (exon 9) |
GGGTGTGGAGCTTTTCCAT |
CCCCTTGCTTCTTTGTTCAG |
1.5 |
60 |
229 |
| SPARC-10 (exon 10) | TCCACTGACTCCTTGGGAAG | GGCAGAACAACAAACCATCC | 1.5 | 60 | 198 |
The promoter (−1 to −318 bp from the transcription initiation site), 5′-untranslated region (the noncoding exon 1), coding regions, exon-intron boundaries, and a portion of the 3′-untranslated region (+1 to +94 bp downstream the stop codon) were covered by the amplimers. In the table, Ta indicates annealing temperature and Bp indicates base pairs.