Geneamp xl pcr kit

Genomic DNA from patient samples was extracted with the AllPrep DNA/RNA Kit (Qiagen) according to the manufacturer’s instructions. PCR was performed with a GeneAmp XL PCR kit (Applied Biosystems) as previously described (23 (link), 24 (link)). The sequences for primers applied to amplify MYD88, CD79A, CD79B, CARD11, and TNFAIP3 are summarized in Supplementary Table S2. The PCR products were visualized by electrophoresis on a 1% agarose gel and ethidium bromide staining. The templates were purified using the QuickStep2 96-well PCR purification Kit (Edge BioSystems) and subsequently sequenced (BigDye sequencing system, Applied Biosystems). Mutations were confirmed on independent PCR products and sequenced from both strands.
Genetic mutations in the B-cell receptor (BCR) pathway have been shown to result in constitutive activity of NFκB, leading to deregulated proliferation and survival signals (23 (link), 25 (link)). In addition, mutations in this pathway are predicted to result in intrinsic resistance to targeted agents such as ibrutinib. Indeed, mutations in CARD11 or TNFAIP3 have been shown to inhibit clinical response to ibrutinib in R/R DLBCL (26 (link)). We therefore performed targeted sequencing of genes in the BCR pathway (MYD88, CD79A, CD79B, CARD11, and TNFAIP3) to understand if mutations abrogated lenalidomide activity.

cDNA was amplified by nested PCR using the GeneAmp XL PCR kit (Applied Biosystems, Foster City, CA). For primary PCR, 5 µL of cDNA was amplified using primers P3 (TATGAYACCCGCTGYTTTGACTC) and P4 (GCNGARTAYCTVGTCATAGCCTC). Secondary PCR conditions were identical to the primary round, except that primer P4 was substituted with primer P5 (GCTAGTCATAGCCTCCGT).This amplification rendered a 260 bp amplicon, which was extracted from 1% agarose by using the QIAquick gel extraction kit (Qiagen, Valencia, CA) and sequenced at the Wadsworth Center’s Applied Genomic Technologies Core Facility.