Table 3.
Amplicon | Primer name | Primer sequences (5′-3′) | Location* | Annealing (°C/s) | Predicted product size (bp) | Genbank Accession No., [Ref] |
---|---|---|---|---|---|---|
cDNA cloning | ||||||
Fragment 1 | dCMAH-cE1aF | CTGTTTTGTGCAGTTTGGCCTCTT | exon 1 (5′-UTR) | *55/60 | 1146 | [30] |
dCMAH-cE9R | TTGTCTGATGGATGGGATTCCACA | exon 9 | NC_006617.3 | |||
Fragment 2 | dCMAH-cE8F | CCTGAGAAGGTTGCTCTAATGA | exon 8 | *55/60 | 925 | NC_006617.3 |
cDMAH-cE15R | TGATCAAGATGTAGCGTCAGTAAAT | exon 15 (3′-UTR) | NC_006617.3 | |||
Fragment 3 | dCMAH-cE1aF | CTGTTTTGTGCAGTTTGGCCTCTT | exon 1 (5′-UTR) | *55/180 | 1852 | [30] |
cDMAH-cE15R | TGATCAAGATGTAGCGTCAGTAAAT | exon 15 (3′-UTR) | NC_006617.3 | |||
SNP discovery | ||||||
Fragment 1 | dCMAH-gE1F | CTCCAGGCTGCCGTCCTTCTA | Promoter | *55/30 | 227 | NC_006617.3 |
dCMAH-gE1R | CAACAGCTTTCAGTTCTTGAAT | intron 1 | NC_006617.3 | |||
Fragment 2 | dCMAH-gE2F | GCCTGGATACTTGGAGGGAGG | intron 1 | *55/30 | 393 | NC_006617.3 |
dCMAH-gE2R | TGATCAGAGAGATGTCCTAAGT | intron 2 | NC_006617.3 | |||
Fragment 3 | dCMAH-gE3F | AGTATTATCTCCTAATGGTTT | intron 2 | *55/30 | 369 | NC_006617.3 |
dCMAH-gE3R | AAGTCCGACTCTACACAGTTT | intron 3 | NC_006617.3 | |||
Fragment 4 | dCMAH-gE4F | TGAGTTGGTGTTGGTCTTAAG | intron 3 | *55/30 | 483 | NC_006617.3 |
dCMAH-gE4R | TCCTAAGAAATTTGCTTAATAG | intron 4 | NC_006617.3 | |||
Fragment | dCMAH-gE5F | TTGAGCATTCTTAGAAGCGAA | intron 4 | *55/30 | 500 | NC_006617.3 |
dCMAH-gE5R | CCTCACTCTGAGTGGTATATA | intron 5 | NC_006617.3 | |||
Fragment 6 | dCMAH-gE6F | AATTCCTTGCTTCTTGATCAACA | intron 5 | *55/30 | 334 | NC_006617.3 |
dCMAH-gE6R | ATCTTAAAGTAACTACCTCATCTAC | intron 6 | NC_006617.3 | |||
Fragment 7 | dCMAH-gE7F | GAAGGATTTCTTTCCAGATGAGC | intron 6 | *55/30 | 254 | NC_006617.3 |
dCMAH-gE7R | CAGATGGCTGTTATCACCCTT | intron 7 | NC_006617.3 | |||
Fragment 8 | dCMAH-gE8F | GGACATAAGTGATGCTTCTCTA | intron 7 | *55/30 | 445 | NC_006617.3 |
dCMAH-gE8R | GGCGCTAAACCACTAAGCCAC | intron 8 | NC_006617.3 | |||
Fragment 9 | dCMAH-gE9F | TTCCTGTGTTAGTCTATCCAT | intron 8 | *55/30 | 386 | NC_006617.3 |
dCMAH-gE9R | AAGCTCCGTGCTCTCAGGCAG | intron 9 | NC_006617.3 | |||
Fragment 10 | dCMAH-gE10F | TAAGTGGATAGGATTGTGAAG | intron 9 | *55/30 | 373 | NC_006617.3 |
dCMAH-gE10R | CCTACAAATAAGGACTTGTCA | intron 10 | NC_006617.3 | |||
Fragment 11 | dCMAH-gE11F | GATAAGAGAACTTTCCTGTAT | intron 10 | *55/30 | 433 | NC_006617.3 |
dCMAH-gE11R | TCCGTGAAGCAGTTGGTAGCG | intron 11 | NC_006617.3 | |||
Fragment 12 | dCMAH-gE12F | CATTGCTATCAATTAAGGCTG | intron 11 | *55/30 | 360 | NC_006617.3 |
dCMAH-gE12R | GTAAAGTAAGGTGTTAACAAT | intron 12 | NC_006617.3 | |||
Fragment 13 | dCMAH-gE13F | AGCCTGTCATATCTACTCCAT | intron 12 | *55/30 | 393 | NC_006617.3 |
dCMAH-gE13R | GAAATGAAGCCCTAATGGTGG | intron 13 | NC_006617.3 | |||
Fragment 14 | dCMAH-gE14F | CAGTATGGAAGCACCATCTCT | intron 13 | *55/30 | 353 | NC_006617.3 |
dCMAH-gE14R | ATGTTCTTGCAATGTTAGCTC | intron 14 | NC_006617.3 | |||
mRNA expression | ||||||
CMAH | dCMAH-cE2F | TGTGCAAGCATCAAGGAGGC | exon 2 | *55/30 | 342 | NC_006617.3 |
dCMAH-cE4R | AGGCAGGACCTATTAACCAG | exon 4 | NC_006617.3 | |||
GAPDH | dGAPDH-cE1/2 | CACAGTCAAGGCTGAGAACG | exon 1/2 | *55/30 | 101 | [37] |
dGAPDH-cE2 | CACCAGCATCACCCCATTT | exon 2 | [37] |
Number of exon is detemined by comaparison between Ac. No. LC382414 and NC_006617.3 in this study
NC_006617.3: Canis lupus familiaris breed boxer chromosome 35, CanFam3.1, whole genome shotgun sequence
d dog, E: exon, F Forward primer, R reverse primer, number exon number, c designed, g designed from genomic sequence in primer name