Point-of-care testing (POCT) diagnostic systems using microfluidic lab-on-a-chip technologies

A fully integrated microdevice and process for forensic short tandem repeat (STR) analysis has been developed that includes sequence-specific DNA template purification, polymerase chain reaction (PCR), post-PCR cleanup and inline injection, and capillary electrophoresis (CE). Fragmented genomic DNA is hybridized with biotin-labeled capture oligos and pumped through a fluidized bed of magnetically immobilized streptavidin-coated beads in microchannels where the target DNA is bound to the beads. The bead–DNA conjugates are then transferred into a 250 nL PCR reactor for autosomal STR amplification using one biotin and one fluorescence-labeled primer. The resulting biotin-labeled PCR products are electrophoretically injected through a streptavidin-modified capture gel where they are captured to form a concentrated and purified injection plug. The thermally released sample plug is injected into a 14 cm long CE column for fragment separation and detection. The DNA template capture efficiency provided by the on-chip sequence-specific template purification is determined to be 5.4% using K562 standard DNA. This system can produce full 9-plex STR profiles from 2.5 ng input standard DNA and obtain STR profiles from oral swabs in about 3 hours. This fully integrated microsystem with sample-in-answer-out capability is a significant advance in the development of rapid, sensitive, and reliable micro-total analysis systems for on-site human identification.

Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification

By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

Multiplex PCR: Critical Parameters and Step-by-Step Protocol

The inventive self-contained apparatus for isolating nucleic acid, cell lysates and cell suspensions from unprocessed samples apparatus, to be used with an instrument, comprises at least one input, and: (i) a macrofluidic component, comprising a chamber for receiving said unprocessed sample from a collection device and at least one filled liquid purification reagent storage reservoir; and (ii) a microfluidic component in communication with said macrofluidic component via at least one microfluidic element, said microfluidic component further comprising at least one nucleic acid purification matrix; and (iii) a drive mechanism on said instrument for driving said liquid purification reagent, through said microfluidic element and said nucleic acid purification matrix, wherein the only inputs to said apparatus are via said chamber and said drive mechanism.

Nucleic acid purification

Forensic DNA typing involves a multi-step workflow. Steps include DNA isolation, quantification, amplification of a set of short tandem repeat (STR) markers, separation of polymerase chain reaction (PCR) products by capillary electrophoresis (CE) and DNA profile analysis and interpretation. With that, the process takes around 10-12h. For several scenarios it may be very valuable to speed up this process and obtain an interpretable DNA profile, suited to search a DNA database, within a few hours. For instance in cases of national security, abduction with danger of life, risk of repetition by a serial perpetrator or when custody time of suspects is limited. By a direct and rapid PCR approach we reduced the total DNA profiling time to 2-3h after which genotyping information for the 10 STR markers plus the amelogenin (AMEL) marker present in the commercially available AmpFlSTR(®) SGM Plus™ (SGM+) profiling kit is obtained. This reduction in time is achieved by using the following elements: (1) the inhibitor tolerant, highly processive Phusion(®) Flash DNA polymerase; (2) a modified, non-adenylated allelic ladder; (3) the quick PIKO(®) thermal cycler system with ultra-thin walled reaction tubes; (4) profile interpretation guidelines with an increased allele calling threshold, modified stutter ratios and marked low-level artefact peaks and (5) regulation of sample input by the use of mini-tapes that lift a limited amount of cell material from swabs or fabrics. The procedure is specifically effective for high level DNA, single source samples such as samples containing saliva, blood, semen and hair roots. Success rates, defined as a complete DNA profile, depend on stain type and surface. Due to the use of tape lifting as the sampling technique, the swab or fabric remains dry and intact and can be analyzed at a later stage using regular procedures. Validation experiments were performed which showed that the protocol effectively instructs researchers unfamiliar with the procedure. We have incorporated direct and rapid PCR in a "DNA-6h" service that can assist police investigations by rapidly deriving DNA information from trace evidence left by a perpetrator, searching the STR profile against a DNA database and reporting the outcomes to police or prosecution.

A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples

The time required for short tandem repeat (STR) amplification is determined by the temperature ramp rates of the thermal cycler,the components of the reaction mix, and the properties of the reaction vessel. Multiplex amplifications in microfluidic biochip-based and conventionaltube-based thermal cyclers have been demonstrated in 17.3 and 19 min, respectively. Optimized 28-cycle amplification protocols generated alleleswith signal strengths above calling thresholds, heterozygous peak height ratios of greater than 0.65, and incomplete nontemplate nucleotide additionand stutter of less than 15%. Full CODIS-compatible profiles were generated using the Profiler Plus ID, COfiler and Identifiler primer sets. PCR per-formance over a wide range of DNA template levels from 0.006 to 4 ng was characterized by separation and detection on a microfluidic electropho-resis system, Genebench-FXTM. The fast multiplex PCR approach has the potential to reduce process time and cost for STR analysis and enablesdevelopment of a fully integrated microfluidic forensic DNA analysis system

Roger Lam

Papers

Fast multiplexed polymerase chain reaction for conventional and microfluidic short tandem repeat analysis.