Solid-phase Reversible Immobilization Research Articles

Automated Purification of DNA Origami with SPRI Beads.

DNA origami synthesis is a well-established technique with wide-ranging applications. In most cases, the synthesized origami must be purified to remove excess materials such as DNA oligos and other functional molecules. While several purification techniques are routinely used, all have limitations, and cannot be integrated with robotic systems. Here the use of solid-phase reversible immobilization (SPRI) beads as a scalable, high-throughput, and automatable method to purify DNA origami is demonstrated. Not only can this method remove unreacted oligos and biomolecules with yields comparable to existing methods while maintaining the high structural integrity of the origami, but it can also be integrated into an automated workflow to purify simultaneously large numbers and quantities of samples. It is envisioned that the SPRI beads purification method will improve the scalability of DNA nanostructures synthesis both for research and commercial applications.

A high-throughput pipeline for DNA/RNA/small RNA purification from tissue samples for sequencing.

High-throughput total nucleic acid (TNA) purification methods based on solid-phase reversible immobilization (SPRI) beads produce TNA suitable for both genomic and transcriptomic applications. Even so, small RNA species, including miRNA, bind weakly to SPRI beads under standard TNA purification conditions, necessitating a separate workflow using column-based methods that are difficult to automate. Here, an SPRI-based high-throughput TNA purification protocol that recovers DNA, RNAand small RNA, calledGSC-modified RLT+ Aline bead-based protocol (GRAB-ALL), which incorporates modifications to enhance small RNA recoveryis presented. GRAB-ALL wasbenchmarkedagainst existing nucleic acid purification workflows and GRAB-ALL efficiently purifies TNA, including small RNA, for next-generation sequencing applications in a plate-based format suitable for automated high-throughput sample preparation.

An SPRI beads-based DNA purification strategy for flexibility and cost-effectiveness

BackgroundCurrent solid-phase reversible immobilization (SPRI) beads technology is widely used in molecular biology due to its convenience for DNA manipulation. However, the high performance commercial SPRI beads have no price advantage over our method. Furthermore, the use of commercially available SPRI beads standards does not provide the flexibility required for a number of specific nucleic acid handling scenarios.ResultsWe report an efficient DNA purification strategy by combining home-made beads-suspension buffer with SPRI beads. The method tests the critical concentrations of polyethylene glycol (PEG) 8000 and beads to maximise recovery. And the composition of the SPRI beads DNA purification system (SDPS) was determined at 20% PEG 8000, 2 M NaCl and 16.3 mM MgCl2, and 1.25 mg/ml beads (1/8th original concentration). Then, we tested the DNA recovery of the SDPS, and the result showed that it was comparable to the control (AMPure XP beads). In the study, we have also developed an adjustment SPRI beads DNA purification system (ASDPS), the volume of ASDPS per reaction is 0.6× reaction volume (beads/samples). The performance of ASDPS is similar to SDPS and the control. But the cost of our methods is only about 1/24th of the control. To further assess its performance, we prepare the DNA-seq libraries to evaluate the yield, library quality, capture efficiency and consistency. We have compared all these results with the performance of the control and confirmed its efficiency.ConclusionWe have proposed an alternative DNA purification approach with great flexibility, allowing researchers to manipulate DNA in different conditions. And ultimately, its application will benefit molecular biology research in the future.

DNA concentration by solid phase reversible immobilization improves its yield and purity, and detection time of E. coli O157:H7 in foods by high resolution melt curve qPCR

For molecular detection of foodborne pathogens to be effective, an efficient technique for bacterial DNA concentration is required. Solid phase reversible immobilization (SPRI) paramagnetic beads can concentrate DNA from foods containing low amounts of bacteria because of their highly specific DNA-binding capacity. This study aimed to develop an upstream bacterial DNA concentration step using SPRI beads and compare its efficacy with immunomagnetic separation (IMS) for the detection of Shiga toxin producing E. coli (STEC) O157:H7 in food samples via a multiplex PCR assay. Food samples were inoculated with STEC O157:H7 at concentrations of 10, 100 and 1000 CFU/mL. The two different concentration methods were compared based on 1) DNA yield 2) DNA purity and 3) detection limit and time of the target pathogen using a high-resolution melt cure multiplex real-time PCR assay for the detection of the major virulence genes, eaeA, stx1 and stx2, in E. coli O157:H7. DNA samples isolated and concentrated by SPRI showed better purity and yield compared to IMS cell concentration. A detection limit of 10 CFU was attained for the majority of inoculated food samples utilizing DNA samples concentrated using SPRI in just 4 h as opposed to 6 or more hours using IMS. SPRI paramagnetic beads in combination with multiplex HRM real-time PCR can be used to rapidly concentrate and detect low concentrations of E. coli O157:H7 in food samples that cannot be detected by conventional culture techniques.

Optimized synthesis of polyacrylic acid-coated magnetic nanoparticles for high-efficiency DNA isolation and size selection.

Solid-phase reversible immobilization (SPRI) bead technology is widely used in molecular biology for convenient DNA manipulation. However, commercial SPRI bead kits lack cost advantages and flexibility. It is, therefore, necessary to develop new and alternative cost-effective methods of on-par or better quality. Herein, an easy and cost-effective method is proposed for synthesizing polyacrylic acid-coated iron oxide nanoparticles (PAA-IONPs) through in situ polymerization at lab scale for high-efficiency nucleic acid extraction and size selection. A design of experiment (DoE) approach was used to investigate the influence of iron oxide nanoparticles (IONPs), acrylic acid (AA) monomer, and sodium dodecyl sulfate (SDS) surfactant amounts on the sizes and carboxyl group densities of PAA-IONPs. Thorough characterization by thermogravimetric analysis (TGA), attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and vibrating sample magnetometry (VSM) highlights the importance of a low starting pH achieved by a high ratio of AA/IONPs, to yield the largest sizes (554 nm) and highest carboxyl group densities (2.13 mmol g-1) obtained in this study. An efficient DNA purification strategy is then presented using homemade beads-suspension buffer and optimized bead concentrations (17% PEG 8000, 2.5 M NaCl, and 3 mg mL-1 PAA-IONPs). This method shows comparable performance to the control (AMPure XP beads) for DNA recovery. An adjustable PAA-IONPs DNA purification system was also developed to be used for DNA-size selection at low DNA amounts (50-100 ng) with a high degree of resolution and recovery. In conclusion, this work offers an optimized PAA-IONPs synthesis protocol and a flexible DNA purification approach that will enable researchers to manipulate DNA under various conditions, holding the significant potential to benefit future molecular biology research and diagnostics.

Optimizing the Use of Solid-Phase Reversible Immobilization Beads for High-Throughput Full-Length 16S rDNA Sequencing Library Construction

Objective: Solid-phase reversible immobilization (SPRI) beads are widely used for high-throughput sequencing library construction to purify and recover nucleic acids. This research was aimed at investigating the effects of SPRI bead ratio, incubation time, and elution time on nucleic acid recovery during full-length 16S rDNA high-throughput sequencing library construction. Methods: The effects of different SPRI bead ratios, incubation times, and elution times were compared for three different initial sample amounts. An L9(33) orthogonal experiment was designed to determine the optimal combination of these factors. Results: The incubation time of three factors including SPRI beads ratio, incubation time, and elution time had a statistically significant effect on the recovery rate for the initial sample amount of 1500 ng and 3000 ng. The orthogonal experiment results indicated that incubation time had the greatest impact among the three factors. Conclusion: Incubation time significantly influences recovery rate in full-length 16S rDNA high-throughput sequencing library construction. The use of 0.8× SPRI beads, 15 minutes of incubation, and 10 minutes of elution resulted in the highest recovery rate. SPRI beads offer a viable method for recovering full-length 16S rDNA amplicons.

Nanopore Whole Transcriptome Analysis and Pathogen Surveillance by a Novel Solid-Phase Catalysis Approach.

The requirement of a large input amount (500 ng) for Nanopore direct RNA‐seq presents a major challenge for low input transcriptomic analysis and early pathogen surveillance. The high RNA input requirement is attributed to significant sample loss associated with library preparation using solid‐phase reversible immobilization (SPRI) beads. A novel solid‐phase catalysis strategy for RNA library preparation to circumvent the need for SPRI bead purification to remove enzymes is reported here. This new approach leverages concurrent processing of non‐polyadenylated transcripts with immobilized poly(A) polymerase and T4 DNA ligase, followed by directly loading the prepared library onto a flow cell. Whole transcriptome sequencing, using a human pathogen Listeria monocytogenes as a model, demonstrates this new method displays little sample loss, takes much less time, and generates higher sequencing throughput correlated with reduced nanopore fouling compared to the current library preparation for 500 ng input. Consequently, this approach enables Nanopore low‐input direct RNA‐seq, improving pathogen detection and transcript identification in a microbial community standard with spike‐in transcript controls. Besides, as evident in the bioinformatic analysis, the new method provides accurate RNA consensus with high fidelity and identifies higher numbers of expressed genes for both high and low input RNA amounts.

Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes

The initial interactions between incoming, pre-replicated virion RNA and host protein factors are important in infection and immunity. Yet currently there are no methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA-host protein interactions. First, host cells are infected with 4-thiouridine (4SU)-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU-labeled viral genomes and interacting proteins from host or virus. The crosslinked RNA binding proteins (RBPs) are purified by solid-phase reversible immobilization (SPRI) beads with protein-denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming virion RNAs are labeled with 4SU, so crosslinking events specifically occur between proteins and pre-replicated virion RNA. Since solid-phase purification under protein-denaturing conditions, rather than sequence-specific nucleic acid purification, is used to pull-down total RNA and crosslinked RBPs, this method facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes ∼7 days and VIR-CLASP takes 1 day.

SPRI Beads-based Size Selection in the Range of 2-10kb.

Application of solid-phase reversible immobilization (SPRI) beads for size selection in molecular biology should be expanded, in light of the property of the beads to accommodate to high MW intervals of DNA fragment size selection, depending on composition of bead-suspension buffer. Here we show how the conventional size selection interval of 150-800 bp be shifted to 1.5-7 Kbp with by adjusting the concentration of NaCl in the stock suspension buffer. The MW capacity of SPRI beads also change when NaCl replaced with other cations and when the concentration of polyethylene glycol (PEG) 8000 is decreased. Testing the limits of SPRI beads revealed cuts as high as 10 Kbp are possible for some salt/PEG combinations of modified SPRI beads-suspension buffers.

Abstract 2807: Accessing the transcriptome of low-abundance tumor-infiltrating lymphocytes with SPRI beads

Abstract Background: Multicolor flow cytometry has provided deep characterization of tumor-infiltrating lymphocytes (TILs). Routine fluorescence-activated cell sorting (FACS) methods discard cells after counting, precluding transcriptome analysis, which is key to understanding the activation states of immune cells. FACS-sorted cells pose 3 problems for transcriptomics: low cell count, cell rupture from high-pressure sort process, and inefficient centrifugation and recovery of cells from large collection volumes. SMART-Seq (TaKaRa/Clontech) is a highly sensitive, reverse transcriptase-based method to amplify low-input RNA that is commonly used to generate RNA-Seq libraries. After FACS, cell RNA can be purified by Qiagen RNeasy or nucleic acid binding solid-phase reversible immobilization (SPRI) magnetic beads. Alternatively, cells can be spun down after FACS then directly lysed as input to the SMART-Seq method. We sought to compare different methods and design a high-throughput protocol to capture the transcriptome from FACS-sorted, rare immune cell types. Methods: A dilution series of 50-5000 human T cells was generated to test efficiency and sensitivity of RNA extraction. We compared RNA extraction yields by column (Qiagen RNeasy Low Input Micro Kit) or SPRI bead (Agencourt RNA-Advanced Cell v2, Beckman Coulter). Direct lysis of cells in SMART-Seq buffer was tested as an alternative to extraction. RNA yields were quantified by High Sensitivity Agilent Bioanalyzer or digital droplet PCR (BioRad). Extracted RNA was reverse transcribed and amplified using SMART-Seq v2 followed by library preparation (Nextera XT DNA kit) and sequencing (NovaSeq Illumina). To obtain TILs, mice were implanted subcutaneously with MC38 tumor cells, then tumors were harvested, disassociated, and FACS sorted to isolate T cells (CD4+, CD8+) and macrophages (yields ranged from 1000-8000 cells). Results: Direct lysis followed by SMART-Seq was found to work well with diluted T cells; however, the method was sensitive to large buffer volumes and was unreliable for small numbers of pelleted, FACS-sorted cells (<5000). SPRI beads provided higher yields than Qiagen columns and were amenable to plate-based automation. RNA-Seq analysis of a T cell dilution series showed high concordance between SPRI and Qiagen columns, although more genes expressed at very low levels were detected in SPRI-purified samples. We used the SPRI bead method to characterize the transcriptome of TILs from implanted mouse tumors. Expected patterns of immune marker expression by immune cell type were observed. Conclusions: Use of SPRI beads to extract RNA from FACS-sorted immune cells was sensitive and reproducible, enabling transcriptomic characterization of <5000 cells. Successful profiling of TILs derived from mouse syngeneic tumors suggests that the method can be used to probe activation states of rare immune cell types in the tumor microenvironment. Citation Format: Aiqing He, Manling Ma-Edmonds, Chan Gao, Vishal Patel, David Galbraith, Yan Zhang, Qi Guo, Heshani DeSilva, Gennaro Dito, Amy Truong, Sunil Kuppasani, Kandasamy Ravi, Omar Jabado. Accessing the transcriptome of low-abundance tumor-infiltrating lymphocytes with SPRI beads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2807.

QsRNA-seq: A protocol for generating libraries for high-throughput sequencing of small RNAs.

Small RNAs (sRNAs) are 20-30 nt long non-coding RNA molecules that regulate essentially all cellular processes. Besides being an intensively studied topic in academic research, sRNAs also hold a promise as clinical biomarkers. While the need for expressional profiling of sRNAs is growing, preparation of sRNA libraries for high-throughput sequencing (HTS) remains technically challenging, due to their small size. The common PAGE-based protocol is time-consuming and inefficient due to material loss, while gel-free protocols generate libraries of insufficient quality. To overcome these shortcomings, we modified the conditions of size-selection by Solid Phase Reversible Immobilization (SPRI) in a way that allows separation of nucleic acids shorter than 100 nt and differing in length by only 20 nt. Implementing the method for preparation of small RNA libraries for HTS resulted in QsRNA-seq, a gel-free, fast and easy-to-perform protocol, amenable to automation, generating very clean libraries that result in high-depth expression data. The protocol also utilizes Unique Molecular Identifiers (UMI) for reduction of library preparation biases and to quantify expression levels. QsRNA-seq provides an excellent solution to the growing needs for small RNA expression profiling for research clinical use.

Finding flies in the mushroom soup: Host specificity of fungus-associated communities revisited with a novel molecular method.

Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology-based identifications, we find a species-rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus-associated interaction webs and communities. Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology-based identifications, we find a species-rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus-associated interaction webs and communities.

Abstract 5351: A simple yet powerful method to purify NGS compatible RNA from formalin fixed paraffin embedded tissues using FormaPure DNA

Abstract Extensive archives of formalin-fixed, paraffin-embedded (FFPE) cancer tissues and next generation sequencing (NGS) capabilities provide rich resources for identifying and validating new cancer biomarkers. However, the ability to conduct FFPE-NGS studies has been limited by poor nucleic acid purification methods that are generally difficult and cumbersome to perform, particularly with RNA. FormaPure DNA was recently launched to provide a simplified workflow to extract DNA from FFPE curls. Its nucleic acid binding chemistry employs Beckman Coulter's proprietary SPRI (solid phase reversible immobilization) technology, which purifies nucleic acids without centrifugation or vacuum manifold. The SPRI technology ensures stronger binding and higher quality purification than other silica-coated bead methods that typically lead to lower quality results due to instances of nucleic acid shearing. Here, we present a modified FormaPure DNA workflow to purify RNA from FFPE samples, offering the flexibility to examine both genomic and transcriptomic roles in cancer biomarkers studies. We show that our extractions yield significantly more RNA (up to 2.5 µg per FFPE curl) and take a fraction of the time to perform compared to other benchmarks. Furthermore, we demonstrate that purified RNA can be used to generate high quality NGS data. Lastly, our extraction method can be automated on a variety of Beckman Coulter’s Biomek liquid handling workstations to offer an integrated solution for high-throughput FFPE-RNA-NGS experiments: RNA from 96 FFPE samples can be purified for NGS in less than 5 hours with minimal hands-on time. Citation Format: Jung H. Doh. A simple yet powerful method to purify NGS compatible RNA from formalin fixed paraffin embedded tissues using FormaPure DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5351. doi:10.1158/1538-7445.AM2017-5351

Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures.

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) as Mycobacterium tuberculosis were successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

Ultrasensitive proteome analysis using paramagnetic bead technology.

In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity-limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub-microgram amounts of material. These data present a first view of developmental stage-specific proteome dynamics in Drosophila at a single-embryo resolution, permitting characterization of inter-individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.

Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples.

Next-generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI-based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post-adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250-bp, paired-end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian-specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing.

96-Well Polycarbonate-Based Microfluidic Titer Plate for High-Throughput Purification of DNA and RNA

We report a simple and effective method for the high-throughput purification of a variety of nucleic acids (NAs) from whole cell lysates or whole blood using a photactivated polycarbonate solid-phase reversible immobilization (PPC-SPRI) microfluidic chip. High-throughput operation was achieved by placing 96 purification beds, each containing an array of 3800 20 microm diameter posts, on a single 3" x 5" polycarbonate (PC) wafer fabricated by hot embossing. All beds were interconnected through a common microfluidic network that permitted parallel access through the use of a vacuum and syringe pump for delivery of immobilization buffer (IB) and effluent. The PPC-SPRI purification was accomplished by condensation of NAs onto a UV-modified PC surface in the presence of the IB comprised of polyethylene glycol, NaCl, and ethanol with a composition dependent on the length of the NAs to be isolated and the identity of the sample matrix. The performance of the device was validated by quantification of the recovered material following PCR (for DNA) or RT-PCR (for RNA). The extraction bed load capacity of NAs was 206 +/- 93 ng for gDNA and 165 +/- 81 ng for TRNA from Escherichia coli. Plate-to-plate variability was found to be 35 +/- 10%. The purification process was fast (<30 min) and easy to automate, and the low cost of wafer fabrication makes it appropriate for single-use applications.

A titer plate-based polymer microfluidic platform for high throughput nucleic acid purification

A 96-well solid-phase reversible immobilization (SPRI) reactor plate was designed to demonstrate functional titer plate-based microfluidic platforms. Nickel, large area mold inserts were fabricated using an SU-8 based, UV-LIGA technique on 150 mm diameter silicon substrates. Prior to UV exposure, the prebaked SU-8 resist was flycut to reduce the total thickness variation to less than 5 mum. Excellent UV lithography results, with highly vertical sidewalls, were obtained in the SU-8 by using an UV filter to remove high absorbance wavelengths below 350 nm. Overplating of nickel in the SU-8 patterns produced high quality, high precision, metal mold inserts, which were used to replicate titer plate-based SPRI reactors using hot embossing of polycarbonate (PC). Optimized molding conditions yielded good feature replication fidelity and feature location integrity over the entire surface area. Thermal fusion bonding of the molded PC chips at 150 degrees C resulted in leak-free sealing, which was verified in leakage tests using a fluorescent dye. The assembled SPRI reactor was used for simple, fast purification of genomic DNA from whole cell lysates of several bacterial species, which was verified by PCR amplification of the purified genomic DNA.

Preparation and characterization of carboxyl-group functionalized superparamagnetic nanoparticles and t he potential for bio-applications

In this work, a method was developed to prepare monodispersed carboxyl-group functionalized magnetic nanoparticles. Nanosized maghemite (y-Fe2O3, 7.0 ± 1.0 nm) was synthesized using thermal co-precipitation method and subsequently coated with functional groups by one-step suspension copolymerization. The Fourier transform infrared spectroscopy study and thermogravitmetric analyses confirmed the successful functionalization of carboxyl groups on the surface of magnetic nanocrystals. The surface chemistry makes it possible for SPRI (solid phase reversible immobilization)-based DNA purification. Thus the nanoparticles were employed to isolate plasmid DNA from bacterial cell culture and the results demonstrated its applicability in DNA preparation.

Continuous Flow Thermal Cycler Microchip for DNA Cycle Sequencing

We report here on the use of a polymer-based continuous flow thermal cycler (CFTC) microchip for Sanger cycle sequencing using dye terminator chemistry. The CFTC chip consisted of a 20-loop spiral microfluidic channel hot-embossed into polycarbonate (PC) that had three well-defined temperature zones poised at 95, 55, and 60 degrees C for denaturation, renaturation, and DNA extension, respectively. The sequencing cocktail was hydrodynamically pumped through the microreactor channel at different linear velocities ranging from 1 to 12 mm/s. At a linear velocity of 4 mm/s resulting in a 36-s extension time, a read length of >600 bp could be obtained in a total reaction time of 14.6 min. Further increases in the flow rate resulted in a reduction in the total reaction time but also produced a decrease in the sequencing read length. The CFTC chip could be reused for subsequent sequencing runs (>30) with negligible amounts of carryover contamination or degradation in the sequencing read length. The CFTC microchip was subsequently coupled to a solid-phase reversible immobilization (SPRI) microchip made from PC for purification of the DNA sequencing ladders (i.e., removal of excess dye-labeled dideoxynucleotides, DNA template, and salts) prior to gel electrophoresis. Coupling of the CFTC chip to the SPRI microchip showed read lengths similar to that obtained from benchtop instruments but did not require manual manipulation of the cycle sequencing reactions following amplification.