Assays for Signal Ends. Ligation-mediated (LM)-PCR assays for 3' Db 2, 5' Jb 2, 5' Db 1, 5' Db 2, and Ja 50 signal ends were carried out as described (1–3). After ligation with the linker oligonucleotide, the primary PCRs were done with the distal template-specific primers 3'Db 2A, 5'Jb 2A, 5'Db 1A, or 5'Db 2A, and the linker-specific primer BW-1H. Five microliters of the primary reaction was used in secondary reactions with the proximal template-specific primer 3'Db 2B, 5'Jb 2B, 5'Db 1B, or 5'Db 2B, and the linker-specific primer BW-1H. Cycling parameters were 95°C for 45 sec, 64°C for 30 sec, and 72°C for 45 sec, for 15 cycles (primary) and 20 cycles (secondary). Primer sequences and oligonucleotide probes were as follows:

3'Db 2A: 5'-TCCCTGGATAGCCTTTCACACTCG-3';

3'Db 2B: 5'-GACCTTGTGAGTCCACTCACATAG-3';

3'Db 2 probe: 5'-ACACCAACTCTCAGCCCA-3';

5'Jb 2A: 5'-CGTTCCCAAGCCAAAAGTGGTATC-3';

5'Jb 2B: 5'-AATTTGAGATCGGCCTCATGCAAG-3';

5'Jb 2 probe: 5'-ACCAGTTCTGGAGGTAGA-3'

5'Db 1A: 5'-GAACAGGGGGTAAAGAGGAAACCC-3';

5'Db 1B: 5'-CATTAGCTCGCATCTTACCACCAC-3';

5'Db 1 probe: 5'-GGTAGACCTATGGGAGGGTC-3';

5'Db 2A: 5'-GATTTACCCAGCTTGAGACTTTTCC-3';

5'Db 2B: 5'-CAGCCCCTCTCAGTCAGACAAACC-3';

5'Db 2 probe: 5'-TGCCACCTGGTCTCCCTGCCCCTGC-3'.

Assays for Db 1-to-Db 2 Coding Joints. Db 1-to-Db 2 coding joints were assayed as described with slight modification (4). The primary PCRs were done with the upstream primer 5'-GGTAGACCTATGGGAGGGTC-3' and downstream primer Db 2R2 (4). Five microliters of the primary reaction was used in secondary reactions with the same upstream primer and Db 2R1 (4). Cycling parameters were 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, for 15 cycles (primary) and 20 cycles (secondary). Db 2R oligonucleotide (4) was used for the hybridization probe.

Assays for Germ-Line Db 1 and Db 2 Levels. The levels of germ-line Db 1 and Db 2 were measured by nested PCR. The primary PCRs used 5' Db 1A, 3' Db 1B or 5' Db 2A, 3' Db 2A primer pairs to amplify germ-line Db 1 and Db 2 sequences, respectively. Five microliters of primary reaction was used in secondary reactions with 5' Db 1B, 3' Db 1B (Db 1) or 5' Db 2B, 3' Db 2B (Db 2) primer pairs. Cycling parameters were 95°C for 1 min, 62°C for 1 min, and 72°C for 3 min, for 15 cycles (primary) and 18 cycles (secondary). Primer sequences were as follows:

5' Db 1A: 5'-CCAGAGGAGCAGCTTATCTGGTGGTTTC-3';

5' Db 1B: 5'-GCCCTCAAGGGGTAGACCTATGGGAGG-3';

3' Db 1A: 5'-ACCTGTAGAACTGTTCACCTCTGGCTC-3';

3' Db 1B: 5'-CCACTGATGGTGGTCTGTTTTATGCAGG-3';

5' Db 2A: 5'-CAGCCCCTCTCAGTCAGACAAACC-3';

5' Db 2B: 5'-CTGCCACCTGGTCTCCCTGCCCCTGC-3';

3' Db 2A: 5'-GACAAAGTGAATTTGAAATCTGAAGGC-3';

3' Db 2B: 5'-CTGTTGTGTTCCAGGTAGCCTCCAATG-3'

1. Schlissel, M., Constantinescu, A., Morrow, T., Baxter, M. & Peng, A. (1993) Genes Dev. 7, 2520–2532.

2. Whitehurst, C., Chattopadhyay, S. & Chen, J. (1999) Immunity 10, 313–322.

3. Leduc, I., Hempel, W. M., Mathieu, N., Verthuy, C., Bouvier, G., Watrin, F. & Ferrier, P. (2000) J. Immunol. 165, 1364–1373.

4. Hempel, W. M., Stanhope-Baker, P., Mathieu, N., Huang, F., Schlissel, M. S. & Ferrier, P. (1998) Genes Dev. 12, 2305–2317.