Microfluidic Devices for Molecular Monitoring of Clonotypic IgH VDJ Signatures and the T(4;14) Translocation in Multiple Myeloma.

Abstract Integrated microfluidic chips offer fast, inexpensive and sensitive detection of molecular characteristics of cancer. PCR was performed using a hybrid polymer/glass chip comprising of wells and channels moulded in polydimethyl siloxane (PDMS) using photolithographic procedures and bonded to glass by a simple rapid prototyping procedure. Fluidic flow was performed by a microprocessor with an in-house built intelligent system for a fully reusable, scalable valving and pumping operation. We successfully performed on-chip PCR with 2 ul of template/PCR mix. Multiple Myeloma (MM) is characterized by a distinct immunoglobulin gene rearrangements, and by IgH translocations that enable unequivocal identification of the MM clone. To detect these molecular signatures, PCR was performed in the central enclosed chamber of a 3-well PCR chip (the other two being the loading and the unloading open wells) using fluorescent-tagged primers, operated using miniaturized user controlled instrumentation. After PCR amplification, product was detected by fragment analysis on a glass capillary electrophoresis (CE) chip using 50–250pl of the amplified product, performed in about 2 minutes. Unlike PCR using plasmid templates, successful PCR using nucleic acid from patient cells required passivation of the PDMS and glass inner surfaces to minimize adsorption of PCR components, and the use of approximately 1–2ng of cDNA template. Amplified products were run through a polymer filled separation channel with size standards to confirm the product size. For two MM patients, using the CE chip, both genomic DNA and IgH VDJ transcripts amplified from individual cells are detectable on-chip with as little as 0.001% of the product (50pL) amplified from one individual MM cell or from groups of MM cells. IgH VDJ product was detectable on the CE chip after a single stage conventional PCR of 30 cycles to amplify genomic DNA from 100 ex-vivo MM cells (100 copies of template). For detection of the single rearranged copy of IgH VDJ in genomic DNA from individual MM cells, a nested PCR strategy was required to amplify sufficient product for detection using either on chip CE or the ABI3100. Compared to analysis on the ABI3100, the gold standard technology, the chip provided approximately 20 fold greater sensitivity for detecting fluorescent product. To further test the microfluidic system, we amplified cDNA from ex-vivo MM cells having the t(4;14)+ translocation to detect hybrid transcripts (IgH-MMSET) on-chip. Cells from MM patients having either the MB4-1 or MB4-3 breakpoint were amplified using the PDMS/glass hybrid chip, and products of the appropriate size were detected using either conventional or on-chip CE. Finally, PCR was performed on an integrated chip that seamlessly incorporates both the PCR and the CE components as a single unit with minimal manual intervention, aiming towards higher levels of on-chip integration. Work is in progress to implement sample processing and cell selection on-chip. This work forecasts automated cost-effective devices able to analyze genetic information in minutes. Real-time detection of complex genetic abnormalities will allow sensitive detection of emerging aggressive variants as disease progresses. This will enable custom tailored therapies that target the genetic vulnerabilities of the malignant clone in each individual patient.

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