Table 1.

SNP genotyping primers for alleles that distinguish between C57BL/6J and C57BL/6N substrains

Ch.	SNP	Temp (°C)	6N size	6J size	Outer size	Forward inner primer (5′-3′)	Reverse inner primer (5′-3′)	Forward outer primer (5′-3′)	Reverse outer primer (5′-3′)
3	rs13477019	55	A-204	T-137	287	AAATGTGCATGCAGTTCTAAACACTA	AACAATATCAATACTAGTTGGATCTGTA	AAATAACTTCAGATTTTCTTCTATAGGAAA	ACTTTACATGATAATTTACCATTGACCT
6	rs13478783	61	G-272	A-201	418	CACTCTGAAAAGGCCCAGGACCAAAA	AGATTGGCCCAGGCCTACCTTTCTTAGTC	CTAGGCCCAAAGAAGAAGAAATGTGGGC	AAGCAGGCGAAGGAGCAAGAGCTAGACT
7	rs13479522	65	G-207	A-253	407	CTGTTGAGAAGCAGGTGCCGGACAAA	TGTGCACTCAGCATTGACGAGAACCAC	CCATCCCATGTGGGAGAGCAAACACTT	CCATGCTTCCAGCCATGATGATAGTGGA
9	rs13480122	65	C-193	T-256	393	AAAGCAGAGAGAGGCTGTACATGCATGTCC	CCAGACCTCGGTGAGGTTGTGGGTTA	CATGTATTCCTGAGGAGAGAGAAGCGGGA	GACTTCAACAGAAACGCCTTTGGAACCA
10	rs13480619	57	C-217	T-259	421	GCTTCCTACTCTTTTGTTTTGTTTTTC	CTAGTTTGAAAAGTCAAACCCAGATTAA	TGCAAGACAAACTCAACTCATACTTTAA	GATGATTCACTACAAGAACAGATCTCAA
10	rs13480759	57	T-268	C-199	414	GGCTTTTGTCTTCTGTAATGTTTCAC	ATTAGAAGACACTTCAGGTTAAGGGAA	ATGTGAAGTCATCGTACACATTTTAGATT	GGTTTATTTTCATTTTCTTGTGTTCTCA
10	rs29359333	65	A-263	G-226	433	AAGGTGGACTACAGTCACAAACAGATTTG	GCATTGTATGTGTGCAATTACACAGGT	ATATGGGGGATGGCTTAGTCAGTAAAAC	TCTAACCCTGTCACATCACAAATTGCTA
11	rs13481014	61	C-252	T-183	379	ATAATCTCCAGTGCATAATGTAGGTGTGTT	GTTAGTCCATTACCCTCTATTTGCGG	TAAAAAATATGCCCTCCTGATTATTCCA	AGTATGTATGTTTCTCTGAGCAGGTTGC
13	rs13481734	61	G-206	A-268	418	AAAGGCAAACACTTGGATCCCATATG	TTTCAAAAAGAATTGATCCCAGAACTGAAT	TGACATTCAGATGCAAAGTGAGTACATGA	CAGAGGGCTAATATCCAAAATGTGTGTG
14	rs31233932	63	T-200	C-298	438	CCCTAATGCTACTTTTTTTCTATTGAGGTT	GTGATAATGAAGAATCGACAAAATTACCTG	GAGAATATGGGCCTCTAGGAAGTTAACA	ACAGAAAGAGAATGAGAATCAAAAGTGC
16	rs4165065	55	C-208	T-179	334	CTACAAACACCCTGAATGCTCATTTT	ATGGAAATATGCCATTTTATTTAATGG	TATTTAACCCTTTGATAGAAAAAGCAGC	AGGTAGGGAAGAAGGTAGGTTTGAGTAG
17	rs13483055	65	C-250	T-213	407	CAGAAAGATCTAATCATTGCCAGGCCAC	TCTGGACCTCCTCTTCTGATAAGGTGCA	GCAGCTTTGGAATTCAGTGACTTTGACAA	GCACAACAGAAACTAAAGCAATCACCAGC

Primers were designed using the Tetra-Primer ARMS PCR method (58,59). In this method, outer primers amplify the region of the SNP and will give a PCR product of the same size (outer size) in both substrains. The forward and reverse inner primers are designed to recognize the different alleles of the SNP and will generate PCR products that differ in size from one another. The table lists the nucleotide present in each substrain at each SNP and the predicted size in base pairs of the PCR product for that substrain (6N size or 6J size). For example, for SNP rs13477019, PCR products of 204 and 287 base pairs would be seen for a mouse that was homozygous for the 6N allele at this SNP, whereas products of 204, 137, and 287 base pairs would be seen if the mouse was heterozygous 6N/6J at this SNP. PCR products were run on a 2% agarose gel and visualized with ethidium bromide. The melting temperatures (temp) used for each four primer reaction are indicated. Ch., chromosome location of the SNP.