An integrated microfluidic device for DNA purification and PCR amplification of STR fragments.
ABSTRACT This work presents the integration of DNA extraction from complex samples and PCR amplification of STR fragments in a valveless, glass microdevice, using commercially available kits and instrumentation. DNA extraction was performed using a microchannel packed with a silica solid phase and a standard syringe pump as a single pressure source driving the extraction process, followed by integrated, online microchip amplification of STR fragments in a total volume of 1.2 microL. Reported characteristics important to this work include the capacity of the device for purification of DNA from a complex biological sample (whole blood) and the timing of DNA elution from the silica solid phase for successful downstream PCR amplification by placement the microdevice into a conventional thermocycler. Potential application of this microdevice to forensic genetic analysis was demonstrated through the preliminary extraction of DNA from semen, followed by an integrated, multiplexed, on-chip amplification that yielded detectable STR amplicons. By utilizing conventional laboratory equipment, the device presented exploits the benefits of microfluidic systems without complex control systems.
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ABSTRACT: Rapid PCR protocols for the amplification of typing short tandem repeat multiplexes were evaluated on 6 different thermal cyclers. Through the use of a faster DNA polymerase coupled with the use of rapid thermal cyclers the amplification cycling times were reduced down to as little as 14 minutes using PCR primers from the commercially available multiplex short tandem repeat typing kit Identifiler. Previously described 2-step and 3-step thermal cycling protocols were evaluated for the 6 thermal cyclers on 95 unique single-source DNA extracts. Capillary electrophoresis characterization of the PCR products indicates good peak balance between loci (median values greater than 0.84), and N minus 4 stutter ratios on averages were 30% to 40% higher than for standard Identifiler PCR conditions. Non-specific amplification artifacts were observed, but were not observed to migrate within the allele calling bins. With the exception of one locus (D18S51) in a single sample, genotyping results were concordant with manufacturer's recommended amplification conditions utilizing standard thermal cycling procedures. Assay conditions were robust enough to routinely amplify 250 pg to 500 pg of template DNA. This work describes the protocols for the rapid PCR amplification of STR multiplexes on various PCR thermal cyclers with the future intent to support validation for typing single-source samples in a databasing laboratory.This article is protected by copyright. All rights reservedElectrophoresis 07/2014; · 3.26 Impact Factor
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ABSTRACT: The generation of short tandem repeat profiles, also referred to as 'DNA typing,' is not currently performed outside the laboratory because the process requires highly skilled technical operators and a controlled laboratory environment and infrastructure with several specialized instruments. The goal of this work was to develop a fully integrated system for the automated generation of short tandem repeat profiles from buccal swab samples, to improve forensic laboratory process flow as well as to enable short tandem repeat profile generation to be performed in police stations and in field-forward military, intelligence, and homeland security settings. An integrated system was developed consisting of an injection-molded microfluidic BioChipSet cassette, a ruggedized instrument, and expert system software. For each of five buccal swabs, the system purifies DNA using guanidinium-based lysis and silica binding, amplifies 15 short tandem repeat loci and the amelogenin locus, electrophoretically separates the resulting amplicons, and generates a profile. No operator processing of the samples is required, and the time from swab insertion to profile generation is 84 minutes. All required reagents are contained within the BioChipSet cassette; these consist of a lyophilized polymerase chain reaction mix and liquids for purification and electrophoretic separation.Profiles obtained from fully automated runs demonstrate that the integrated system generates concordant short tandem repeat profiles. The system exhibits single-base resolution from 100 to greater than 500 bases, with inter-run precision with a standard deviation of +/-0.05 - 0.10 bases for most alleles. The reagents are stable for at least 6 months at 22[degree sign]C, and the instrument has been designed and tested to Military Standard 810F for shock and vibration ruggedization. A nontechnical user can operate the system within or outside the laboratory. The integrated system represents the first generation of a turnkey approach to short tandem repeat profiling and has the potential for use in both the field (for example, police booking stations, the battlefield, borders and ports) and the forensic laboratory.Investigative genetics. 08/2013; 4(1):16.
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ABSTRACT: Since the emergence of lab-on-a-chip technology, a variety of chemical and biochemical assays were successfully implemented on microdevice platforms. Among the chip-based applications, genetic analysis based on the polymerase chain reaction (PCR) has been extensively developed in order to accomplish the goal of cheap, rapid, high-throughput, and point-of-care DNA testing. We are summarizing here several formats of the miniaturized PCR systems including the integration of units for sample pretreatment and downstream analytical detection. The various sections cover (a) miniaturized PCR systems, (b) integrated sample pretreatment-PCR microsystems, (c) integrated PCR-detection microsystems, and (d) integrated sample pretreatment-PCR-detection microsystems. Respective microdevices were successfully introduced recently in the form of a fully integrated microsystem for genetic analysis with sample-in-answer-out capability. Contains 120 references. Figure ᅟMicrochimica Acta · 3.43 Impact Factor