PCR conditions
| First Round of PCR | |||||
| Primer | FRI VH | FRI VH | FRI Vλ | Leader VH | Leader VH3,4 |
| Combination | FRI Vκ | FRI Vλ | 3′Jλ | FRI Vκ | Leader Vλ3 |
| 3′JH, 3′Jκ | 3′JH, 3′Jλ | 3′JH, 3′Jκ | 3′JH, 3′Jλ | ||
| Primer (nmol/L each) | 6.9 | 14.3 | 22.0 | 40.0 | 19.0 |
| dNTP (μmol/L each) | 100 | 200 | 100 | 200 | 100 |
| MgCl2 (mmol/L) | 2.5 | 2.0 | 1.5 | 2.0 | 2.5 |
| Annealing temp. | 59°C | 60°C | 59°C | 61°C | 59°C |
| Second Round of PCR | |||||
| Primer (nmol/L each) | 125 | 125 | 125 | 125 | 125 |
| dNTP (μmol/L each) | 100 | 100 | 100 | 200 | 100 |
| MgCl2 (mmol/L) | VH1-6: 1.5 Vκ1-4: 2.5 | 1.5 | 1.5 | VHL1,5: 1.5 VHL2,3: 2.0 VHL4: 3.0 Vκ1-4: 2.5 | VHL4: 3.0 VλL3: 1.5 |
| Annealing temperature | 61°C: VH1,2,5/6 Vκ1-4 65°C: VH3,4 | 61°C: VH1,2,5/6 65°C: VH3,4 63°C: Vλ1-4,6-9 | 63°C | 63°C: VHL1-5 61°C: Vκ1-4 | 63°C |
| Cells/Samples Analyzed | |||||
| Patient 1 | |||||
| HRS-like | 22 | 8 | 7 | 13 | |
| B-CLL | 22 | 6 | 5 | 29 | |
| Patient 2 | |||||
| HRS-like | 5 | 28 | |||
| B-CLL | 4 | 24 | 8 | ||
| Patient 3 | |||||
| HRS-like | 16 | ||||
| B-CLL | 16 |
| First Round of PCR | |||||
| Primer | FRI VH | FRI VH | FRI Vλ | Leader VH | Leader VH3,4 |
| Combination | FRI Vκ | FRI Vλ | 3′Jλ | FRI Vκ | Leader Vλ3 |
| 3′JH, 3′Jκ | 3′JH, 3′Jλ | 3′JH, 3′Jκ | 3′JH, 3′Jλ | ||
| Primer (nmol/L each) | 6.9 | 14.3 | 22.0 | 40.0 | 19.0 |
| dNTP (μmol/L each) | 100 | 200 | 100 | 200 | 100 |
| MgCl2 (mmol/L) | 2.5 | 2.0 | 1.5 | 2.0 | 2.5 |
| Annealing temp. | 59°C | 60°C | 59°C | 61°C | 59°C |
| Second Round of PCR | |||||
| Primer (nmol/L each) | 125 | 125 | 125 | 125 | 125 |
| dNTP (μmol/L each) | 100 | 100 | 100 | 200 | 100 |
| MgCl2 (mmol/L) | VH1-6: 1.5 Vκ1-4: 2.5 | 1.5 | 1.5 | VHL1,5: 1.5 VHL2,3: 2.0 VHL4: 3.0 Vκ1-4: 2.5 | VHL4: 3.0 VλL3: 1.5 |
| Annealing temperature | 61°C: VH1,2,5/6 Vκ1-4 65°C: VH3,4 | 61°C: VH1,2,5/6 65°C: VH3,4 63°C: Vλ1-4,6-9 | 63°C | 63°C: VHL1-5 61°C: Vκ1-4 | 63°C |
| Cells/Samples Analyzed | |||||
| Patient 1 | |||||
| HRS-like | 22 | 8 | 7 | 13 | |
| B-CLL | 22 | 6 | 5 | 29 | |
| Patient 2 | |||||
| HRS-like | 5 | 28 | |||
| B-CLL | 4 | 24 | 8 | ||
| Patient 3 | |||||
| HRS-like | 16 | ||||
| B-CLL | 16 |
HRS indicates HRS.
Combinations of primers: FRI family-specific primers forVH,Vκ, andVλ genes; VH leader (VHL) region family-specific primers; and VHL3, VHL4, and VλL3 primers specific for the leader regions of VH3, VH4, andVλL3 genes, respectively.
The basic PCR program in the first round of PCR was: 1 cycle for 2 minutes at 95°C, 80°C hold (addition of enzyme); 30 seconds at 59°C, 60°C, or 61°C; and 1 minute at 72°C. This was followed by 34 cycles for 1 minute at 95°C; 30 seconds at 59°C, 60°C, or 61°C; and 1 minute at 72°C, followed by 5 minutes at 72°C. The basic PCR program in the second round of PCR was: 1 cycle for 2 minutes at 95°C, 80°C hold (addition of enzyme); 30 seconds at 61°C, 63°C, or 65°C; and 1 minute at 72°C. This was followed by 44 cycles of 1 minute at 95°C; 30 seconds at 61°C, 63°C, or 65°C; and 1 minute at 72°C, followed by 5 minutes at 72°C.
In the second round of PCR, a 1.5-μL aliquot from the first round was further amplified with the same V gene primers in separate reactions and internal primer sets for the J gene segments.8 12-14 During this second round, each primer was used at a concentration of 125 nmol/L, with the exception of the JH primers, which were used at a concentration of 31.3 nmol/L. In the first round of amplification, 1.5 units of enzyme mix (Expand, Boehringer Mannheim) was added; in the second round, 1.25 units of Taq DNA polymerase (Gibco BRL, Karlsruhe, Germany) was added.