Primer sequences
This page provides genetic testing details for anyone interested in performing genetic tests on their own. These details assume that you have experience in molecular biology.
Keep in mind that if an animal tests negative for a recessive morph, there is always a tiny possibility that the animal carries a novel variant in the causative gene.
PCR
We typically use the DNA polymerase OneTaq, which is available from New England BioLabs. DNA polymerases from other manufacturers will likely also work. Below is our recipe for PCR using OneTaq.
4 uL 5X OneTaq Buffer
0.4 uL 10 mM dNTPs
2 uL Forward primer, 2 uM
2 uL Reverse primer, 2 uM
1 uL genomic DNA, 100 ng/uL
0.1 uL OneTaq
10.5 uL water
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20 uL total volume
Thermocycling conditions:
94C 2 min
30 cycles:
94C 30 sec
57C 30 sec (or annealing temp specified below)
68C 1 min
68C 5 min
Albino & Toffee/Candy
Notes: 1) There are two mutations for Albino. Some Het Albinos have one mutation. Some Het Albinos have the other mutation. This test only identifies one of the mutations. If an animal tests negative using this test, the animal might still be Het Albino if it carries the other mutation.
2) The Albino and Toffee/Candy mutations are close together in the genome and are genotyped using the same test.
Primers:
p242F 5'- GCCATTGTAGCTTCTTACCACTC - 3'
p242R 5'- TTCcAGTCCATATACaAGATATCCAA - 3'
Annealing temperature: 57C
Perform Sanger sequencing of the PCR product with p242F
Wildtype allele: ...ACCATGCCTTCGTGG[A]
Albino (D394L) allele: ...ACCATGCCTTCGTGG[G]
Wildtype allele: ...TTCAGCCAACGATC[C]
Toffee/Candy (P384L) allele: ...TTCAGCCAACGATC[T]
Ultramel
Note: There are two mutations for Ultramel. Some Het Ultramels have one mutation. Some Het Ultramels have the other mutation. This test only identifies one of the mutations. If an animal tests negative using this test, the animal might still be Het Ultramel if it carries the other mutation.
Primers:
p18F 5'- GCTCTTTTCTCTAAGTCTGACCTC - 3'
p18R 5'- TCTTGTCCCACAAAAGGATTT - 3'
Annealing temperature: 57C
Perform Sanger sequencing of the PCR product with p18F
Wildtype allele: ...GCACAGAAGGTGGTCCAATTAGAC[G]
Ultramel allele: ...GCACAGAAGGTGGTCCAATTAGAC[A]
Lavender Albino
Primers:
p217F 5' - GGAGAGAGAATCCAACCCTTG - 3'
p166R 5' - TGGGTGGCAAACAATCATAA - 3'
p188R 5' - CAAAGACCATTGTCCATTTCC - 3'
Annealing temperature: 57C
Perform PCR with all three primers simultaneously. The wildtype allele produces a 429-bp product. The Lavender Albino allele produces a 349-bp product. Heterozygotes produce both products. Bands sizes are shown below.
Wildtype allele: 429-bp product
Lavender Albino allele: 349-bp product
Piebald
Primers:
p364F 5'- TGCAACTCAAAGGGAACAAA - 3'
p364R 5'- GAGCAACATAATTTCCCAAGG - 3'
Annealing temperature: 57C
Perform Sanger sequencing of the PCR product with p364R
Wildtype allele: ...TGAGATTATGGTTGTCCTTTTTTTGTCTCTCTTTTGCCATTGCTC[G]
Piebald allele: ...TGAGATTATGGTTGTCCTTTTTTTGTCTCTCTTTTGCCATTGCTC[A]
Yellowbelly Complex
Note: We have encountered mix-ups among animals in the Yellowbelly Complex. Some animals described a Super Stripe are actually Puma and vice versa. Some animals described as Puma are actually Ivory. Some animals described as Highway are actually Freeway and vice versa. Some animals described as Yellowbelly are actually Spark. Mix-ups tend to be more common among animals with additional genes.
If your animal tests negative for a mutation in the Yellowbelly Complex, you may want to consider the possibility that your animal carries a different mutation in the Yellowbelly Complex.
Spark & Gravel
Note: The Spark and Gravel mutations are close together in the genome and are genotyped using the same test.
Primers:
p381F 5'- CACCATAATGCTTAACACACACAA - 3'
p381R 5'- TCATAGCAATGTGATAAACCCACT - 3'
Annealing temperature: 57C
Perform Sanger sequencing of the PCR product with p381F
Wildtype allele: ...TTCTACATCCTCATTGCTTT[G]
Spark allele: ...TTCTACATCCTCATTGCTTT[C]
Wildtype allele: ...CCCATTAATGTGTATAA[G]
Gravel allele: ...CCCATTAATGTGTATAA[A]
Asphalt
Primers:
p259F 5'- GGCAGGAAAACTGCTCGATA - 3'
p260R 5'- CAAACCAAAGTCCCAGCATC - 3'
Annealing temperature: 57C
Perform Sanger sequencing of the PCR product with p259F
Wildtype allele: ...AAATTTAAGAACTGCTTTCA[G]
Asphalt allele: ...AAATTTAAGAACTGCTTTCA[A]
Yellowbelly
Note: There are two mutations for Yellowbelly. Some Yellowbelly animals have one mutation. Other Yellowbelly animals have the other mutation. Ivory animals can have two copies of one mutation or one copy of each mutation. Both mutations can be identified at the same time using this test.
Primers:
p257F 5'- GGTTCCCAGTCTCTGCACAT - 3'
p257R 5'- TTCAACCAACGCACATCATT - 3'
Annealing temperature: 57C
Digest the PCR product with restriction enzyme BsrI (65C). Each of Yellowbelly mutations destroy a cut site for BsrI. Bands sizes are shown below.
Specter
Primers:
p258F 5'- ATGGAGTGGACAGGATGGAA - 3'
p258R 5'- CCAGCTAGGTGGGGTAGACA - 3'
Annealing temperature: 57C
Digest the PCR product with restriction enzyme SmaI (25C). The Specter mutation each creates a cut site for SmaI. Bands sizes are shown below.
