Automated and Integrated System for High-Throughput DNA Genotyping Directly from Blood

Abstract

An automated and integrated system for DNA typing directly from blood samples has been developed. The multiplexed eight-array system is based on capillary microfluidics and capillary array electrophoresis. Three short-tandem-repeat loci, vWA, THO1, and TPOX, are coamplified simultaneously in a fused-silica capillary by a hot-air thermocycler. Blood is directly used as the sample for polymerase chain reaction (PCR) without any pretreatment. Modifications of standard protocols are necessary for direct PCR from blood. A programmable syringe pump plus a set of multiplexed liquid nitrogen freeze/thaw switching valves are employed for liquid handling in the fluid distribution network. The system fully integrates sample loading, PCR, addition of an absolute standard, on-line injection of sample and standards, separation and detection. The genotypes from blood samples can be clearly identified in eight parallel channels when the electropherograms are compared with that of the standard allelic ladder by itself. Regeneration and cleaning of the entire system prior to subsequent runs are also integrated into the instrument. The instrumentation is compatible with future expansion to hundreds of capillaries to achieve even higher throughput.

High sensitivity PCR assay in plastic micro reactors

“…Considering that M13/pUC is a well established cloning vector and the assay was carried out in a buffer environment, the prospective inhibition from a matrix of 'real' sample (blood sample and/or with whole target cells) was not present. The PC plastic serpentine channel PCR micro reactor discussed in this study held a maximum volume of 40 ml and demonstrated a high sensitivity of amplifying 10 E. coli cells (50 fg DNA) from 2% blood sample, as compared to on-chip (glass-silicon) amplification of ng amounts of WBC DNA obtained by filtration from a sample containing 3 ml of blood (30) and/or PCR from a 1% blood 31 sample in fused-silica capillaries. Although 40 ml is not commonly considered a very small volume, this plastic PCR micro reactor was designed and fabricated with the intent of total on-chip integration.…”

“…37,38 A high sensitivity amplification from a blood sample in a plastic micro PCR reactor or chip had not been demonstrated to date. An automated and integrated system for high-throughput DNA genotyping directly from blood was reported by Zhang et al 31 Using a hot air thermocycler, samples containing whole blood were directly amplified in a fused-silica capillary and only one concentration (1%) of blood sample PCR was demonstrated, providing ng amounts of DNA as the starting genomic material. In another microchip module for blood sample preparation and amplification, Wilding's group demonstrated direct capture of WBC cells in a glass-silicon microchip with a series of micro filters, from 3 ml of whole human blood, and subsequent amplification of human coagulation factor V from WBC DNAs in the same microchip, 30 in which > 10 ng WBC genomic DNA was used as initial template.…”

“…Therefore, the initial template of human WBC DNA concentration from 3 ml of human whole blood was > 10000 cells ( > 10 ng DNA). An automated and integrated system for DNA genotyping directly from blood was reported by Zhang et al 31 With addition of formamide, three short-tandem-repeat loci were amplified in fused-silica capillaries, directly from about 1% blood sample providing ng range of genomic DNA as the starting material.…”

A microfluidic system for high-speed reproducible DNA sizing and quantitation

“…Under optimized conditions, separations more than two orders of magnitude faster than traditional slab gel electrophoresis can be performed [3]. CE in linear sieving matrices can be easily automated and parallel capillary systems which combine the speed of analysis with a high overall throughput are currently being used for genetic analysis [4,5].…”

Ultrafast detection of microsatellite repeat polymorphism in endothelin 1 gene by electrophoresis in short capillaries

“…Although CE separations are faster than those of slab gel electrophoresis, a limitation has been that only one sample is processed at a time. CE in an array of up to 96 parallel capillaries represents another way to increase the analysis throughput [4,21,23,25,32,33]. Further miniaturization of the CE system and its integration with sample preparation procedures has been realized on micromachined chips [2,3,6,8,9].…”