Dataset Information

Reversible Oligonucleotide Chain Blocking Enables Bead Capture and Amplification of T-Cell Receptor ? and ? Chain mRNAs.

ABSTRACT: Next-generation sequencing (NGS) has proven to be an exceptionally powerful tool for studying genetic variation and differences in gene expression profiles between cell populations. However, these population-wide studies are limited by their inability to detect variation between individual cells within a population, inspiring the development of single-cell techniques such as Drop-seq, which add a unique barcode to the mRNA from each cell prior to sequencing. Current Drop-seq technology enables capture, amplification, and barcoding of the entire mRNA transcriptome of individual cells. NGS can then be used to sequence the 3'-end of each message to build a cell-specific transcriptional landscape. However, current technology does not allow high-throughput capture of information distant from the mRNA poly-A tail. Thus, gene profiling would have much greater utility if beads could be generated having multiple transcript-specific capture sequences. Here we report the use of a reversible chain blocking group to enable synthesis of DNA barcoded beads having capture sequences for the constant domains of the T-cell receptor ? and ? chain mRNAs. We demonstrate that these beads can be used to capture and pair TCR? and TCR? sequences from total T-cell RNA, enabling reverse transcription and PCR amplification of these sequences. This is the first example of capture beads having more than one capture sequence, and we envision that this technology will be of high utility for applications such as pairing the antigen receptor chains that give rise to autoimmune diseases or measuring the ratios of mRNA splice variants in cancer stem cells.

SUBMITTER: Hanson WM

PROVIDER: S-EPMC5249220 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Publications

Reversible Oligonucleotide Chain Blocking Enables Bead Capture and Amplification of T-Cell Receptor α and β Chain mRNAs.

Hanson W Miachel WM Chen Zhe Z Jackson Laurie K LK Attaf Meriem M Sewell Andrew K AK Heemstra Jennifer M JM Phillips John D JD

Journal of the American Chemical Society 20160801 35

Next-generation sequencing (NGS) has proven to be an exceptionally powerful tool for studying genetic variation and differences in gene expression profiles between cell populations. However, these population-wide studies are limited by their inability to detect variation between individual cells within a population, inspiring the development of single-cell techniques such as Drop-seq, which add a unique barcode to the mRNA from each cell prior to sequencing. Current Drop-seq technology enables c ...[more]

PMID: 27478996

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Project description:Porous agarose microbeads, with high surface to volume ratios and high binding densities, are attracting attention as highly sensitive, affordable sensor elements for a variety of high performance bioassays. While such polymer microspheres have been extensively studied and reported on previously and are now moving into real-world clinical practice, very little work has been completed to date to model the convection, diffusion, and binding kinetics of soluble reagents captured within such fibrous networks. Here, we report the development of a three-dimensional computational model and provide the initial evidence for its agreement with experimental outcomes derived from the capture and detection of representative protein and genetic biomolecules in 290 ?m porous beads. We compare this model to antibody-mediated capture of C-reactive protein and bovine serum albumin, along with hybridization of oligonucleotide sequences to DNA probes. These results suggest that, due to the porous interior of the agarose bead, internal analyte transport is both diffusion and convection based, and regardless of the nature of analyte, the bead interiors reveal an interesting trickle of convection-driven internal flow. On the basis of this model, the internal to external flow rate ratio is found to be in the range of 1:170 to 1:3100 for beads with agarose concentration ranging from 0.5% to 8% for the sensor ensembles here studied. Further, both model and experimental evidence suggest that binding kinetics strongly affect analyte distribution of captured reagents within the beads. These findings reveal that high association constants create a steep moving boundary in which unbound analytes are held back at the periphery of the bead sensor. Low association constants create a more shallow moving boundary in which unbound analytes diffuse further into the bead before binding. These models agree with experimental evidence and thus serve as a new tool set for the study of bioagent transport processes within a new class of medical microdevices.

Dataset Information

Reversible Oligonucleotide Chain Blocking Enables Bead Capture and Amplification of T-Cell Receptor ? and ? Chain mRNAs.

Publications

Reversible Oligonucleotide Chain Blocking Enables Bead Capture and Amplification of T-Cell Receptor α and β Chain mRNAs.

Similar Datasets

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