In order to amplify using FR2/LJH primers, in the first PCR 50 ng genomic DNA were used and the reaction mix contained 1× PCR buffer, 200 µM 2′-deoxynucleosides 5′-triphosphate (dNTPs), 2 µM primers, 2 mM MgCl2, 0·001% gelatin and 1·5 U Taq DNA polymerase. The PCR conditions were initial denaturation at 95°C

for 7 min followed by 40 cycles of the following parameters: denaturation, 94°C for 45 s; annealing, 50°C for 30 s; and extension, 72°C for 45 s. For the second round the reaction mixture contained 1 µl of the first PCR product and primers FR2 and VLJH. The cycling protocols to FR3/LJH were the same as FR2, with the exception of the annealing temperature (56°C). To amplify the Fr1c/JH1–6 primers, Sirolimus chemical structure we employed the same reaction mix described above without gelatin and

supplemented with 10% dimethylsulphoxide (DMSO), 1·25 U of Taq DNA polymerase and 50 ng of genomic DNA. The PCR conditions were the same as FR2, with the exception selleck kinase inhibitor of 35 cycles and annealing temperature of 60°C. Samples in which DNA amplification was not clear were reamplified using the following specific primers: one directed to the FR1 region and the other to the JH region. PCR to amplify the GAPDH gene was performed under standard conditions, with the exception of an annealing temperature of 55°C. The specific primers are indicated in Table 2 and the samples were amplified as described above. Bcl-2/JH translocation was analysed by a modified PCR–enzyme-linked immunosorbent assay (ELISA) technique (PharmaGen, Madrid, Spain), using primers directed to the major breakpoint region (mbr) and minor Montelukast Sodium breakpoint region (mcr) of the bcl-2 oncogene coupled with LJH

primer as indicated in Table 2[21]. Briefly, the PCR reactions were performed in similar conditions as described above, using 2′-deoxyuridine 5′-triphosphate (dUTP) digoxygenin instead of thymidine triphosphate (dTTP) and 100 ng of genomic DNA at an annealing temperature of 60°C. The amplified product was hybridized to a biotin-labelled probe and quantified by ELISA, according to the manufacturer’s instructions. The PCR reaction was performed under standard conditions, as described above, under the following amplification conditions: initial denaturation at 95°C for 7 min followed by 30 cycles using the following parameters: denaturation, 94°C for 45 s; annealing, 56°C for 45 s; and extension, 72°C for 110 s. The PCR products were analysed on 3% agarose gels using the FR1c/JH1–6 or FR2/LJH-VLJH amplification protocol or 8% polyacrylamide gels using the FR3/LJH amplification protocol. Gels were photographed under ultraviolet light after staining with ethidium bromide or silver nitrate staining. To determine the sensitivity of our IgH PCR method, we prepared serial 10-fold dilutions of the LM cell line (lymphoblastic lymphoma) in normal peripheral blood mononuclear cells (PBMC). For this purpose, 100–105 clonal B lymphocytes from the LM cell line were diluted with 105 PBMC.