Table 1 Primers and Conditions for Sequencing HLA-H, β2 -microglobulin, and Calreticulin
From: HLA-H and Associated Proteins in Patients with Hemochromatosis
Gene | PCR primers | Annealing temperature | Sequencing primers | |
|---|---|---|---|---|
HLA-H | Exon 1 | 5′-CATTGCGAAGCTACTTTCCC-3′ | 56°C | PCR primers |
5′-AGTTTCGATTTTTCCACCCC-3′ | ||||
Exon 2a | 5′-acatggttaaggcctgttgc-3′ | 55°C | PCR primers | |
5′-GCCACATCTGGCTTGAAATT-3′ | ||||
Exon 2b | 5′-CATGAGAGTCGCCGTGTG-3′ | 55°C | PCR primers | |
5′-cagctgtttccttcaagatgc-3′ | ||||
Exon 3a | 5′-tttggttgcagttaacaaggc-3′ | 55°C | PCR primers | |
5′-GAATCTTGTGCCTTTCCCAC-3′ | ||||
Exon 3b | 5′-GGATTGGAGAGCAGCAGAAC-3′ | 56°C | PCR primers | |
5′-aaactccaaccagggattcc-3′ | ||||
Exon 4 | 5′-tggcaagggtaaacagatcc-3′ | 58°C | PCR primers | |
5′-ctcaggcactcctctcaacc-3′ | ||||
Exon 5 | 5′-gcctgaggaggtatatggc-3′ | 58°C | PCR primers | |
5′-gagacttcccccttgttcct-3′ | ||||
Exon 6 | 5′-tgggtgaatgaggaaaataagg-3′ | 55°C | PCR primers | |
5′-ctagggatcaccggcatg-3′ | ||||
β2-microglobulin | Promoter-exon 1 | 5′-tcctgattggctgggcacgc-3′ | 63°C | PCR primers |
5′-cagagcgggagggtaggaga-3′ | ||||
Exon 2 | 5′-accctggcaatattaatgtgtc-3′ | 59°C | PCR primers | |
5′-tacacaactttcagcagcttac-3′ | ||||
Exon 3 | 5′-ttttagacatttgttagtacatgg-3′ | 58°C | PCR primers | |
5′-ctgtaggattcttctttccctg-3′ | ||||
Calreticulin cDNA | Exons 1–3 | 5′-CCGTCCGTCACTGCAGAGC-3′ | 60°C | 5′-CAGAGCCGCTGCCGGAGGGT-3′ |
5′-ACAGCTTCACATAGCCGCC-3′ | 5′-CCCACAGTCGATGTTCTGCT-3′ | |||
Exons 3–5 | 5′-CACGCTTTTATGCTCTGTCG-3′ | 58°C | 5′-TGTCGGCCAGTTTCGAGCCT-3′ | |
5′-AGTCCTCAGGCTTGGAGTC-3′ | 5′-GTGGGATCATCGATCTTGGC-3′ | |||
Exons 5–8 | 5′-ACGATTGGGACTTCCTGCC-3′ | 58°C | 5′-CCCAAGAAGATAAAGGATCCT-3′ | |
5′-GCCTCTGCTCCTCGTCCTG-3′ | 5′-CTCGTCCTGTTTGTCCTTCA-3′ | |||
Exons 8–9 | 5′-GGCACTTGGATCCACCCAG-3′ | 57°C | 5′-GGAGTTTGGCAACGAGACGT-3′ | |
5′-TTGGCGCGGCAAGCTCTG-3′ | 5′-TCTGCGGCAGGAGCGCTCAG-3′ | |||
Calreticulin gDNA | Promoter | 5′-ggtcccggtcccgcgtgg-3′ | 60°C | |
5′-cgcggccctttaaaacgac-3′ | 5′-tccggcagcggctctgcagt-3′ | |||
Exons 1–5 | 5′-CCGTCCGTCACTGCAGAGC-3′ | 60°C | 5′-CAGAGCCGCTGCCGGAGGGT-3′ | |
5′-ACAGCTTCACATAGCCGCC-3′ | 5′-CCCACAGTCGATGTTCTGCT-3′ | |||
5′-CACGCTTTTATGCTCTGTCG-3′ | ||||
5′-GTGGGATCATCGATCTTGGC-3′ | ||||
Exons 5–7 | 5′-ACGATTGGGACTTCCTGCC-3′ | 59°C | PCR primers | |
5′-ctcacCTGCCAGAGGTCC-3′ | ||||
or | ||||
5′-tttgagtcagactcacctgg-3′ | ||||
Exons 8–9 | 5′-GGCACTTGGATCCACCCAG-3′ | 57°C | 5′-GGAGTTTGGCAACGAGACGT-3′ | |
or | ||||
5′-ggtgttccttgtcttctctg-3′ | ||||
5′-TTGGCGCGGCAAGCTCTG-3′ | 5′-TCTGCGGCAGGAGCGCTCAG-3′ | |||
5′-ggtgttccttgtcttctctg-3′ |