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Multiplexed programmable release of captured DNA.

ABSTRACT: Nucleic-acid hybridization is widely used for the specific capture of complementary sequences from complex samples. It is useful for both analytical methodologies, such as array hybridization (e.g. transcriptome analysis, genetic-variation analysis), and preparative strategies such as exome sequencing and sequence-specific proteome capture and analysis (PICh, HyCCAPP). It has not generally been possible to selectively elute particular captured subsequences, however, as the conditions employed for disruption of a duplex can lack the specificity needed to discriminate between different sequences. We show here that it is possible to bind and selectively release multiple sets of sequences by using toehold-mediated DNA branch migration. The strategy is illustrated for simple mixtures of oligonucleotides, for the sequence-specific capture and specific release of crosslinked yeast chromatin, and for the specific release of oligonucleotides hybridized to DNA microarrays.

SUBMITTER: Kennedy-Darling J

PROVIDER: S-EPMC4218743 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Multiplexed programmable release of captured DNA.

Kennedy-Darling Julia J Holden Matthew T MT Shortreed Michael R MR Smith Lloyd M LM

Chembiochem : a European journal of chemical biology 20140826 16

Nucleic-acid hybridization is widely used for the specific capture of complementary sequences from complex samples. It is useful for both analytical methodologies, such as array hybridization (e.g. transcriptome analysis, genetic-variation analysis), and preparative strategies such as exome sequencing and sequence-specific proteome capture and analysis (PICh, HyCCAPP). It has not generally been possible to selectively elute particular captured subsequences, however, as the conditions employed fo ...[more]

PMID: 25157426

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Project description:Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.

Dataset Information

Multiplexed programmable release of captured DNA.

Publications

Multiplexed programmable release of captured DNA.

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