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Abstracts of Oak Ridge Posters |
1 Cepheid, 1190 Borregas Ave., Sunnyvale, CA 94089
aauthor for correspondence: fax 408-541-4191, e-mail ching{at}cepheid.com
The human genome contains microsatellite sequences, consisting of 2- to 5-bp repeats, randomly distributed among the chromosomes. These sequences, also called short tandem repeats (STRs), have been demonstrated as important markers for disease diagnostics, genetic mapping, and human identification. The list of human hereditary diseases associated with the unusual expansion or deletion of specific microsatellite loci continues to increase. The two most understood diseases of this type are the CGG repeats in fragile-X syndrome (1) and the CAG repeats in Huntington disease (2). However, other diseases, such as cancer of the colon, head, neck, gastrointestinal track, urinary bladder, liver, lung, breast, and leukocytes [white blood cells (WBCs)], have been shown to be linked to microsatellite instability (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14).
The growing importance of microsatellite markers in diagnostics has motivated us to develop rapid, automated devices to integrate sample preparation, PCR, and capillary electrophoresis (CE) analysis. The work presented here demonstrates an efficient filtration cartridge (15) to automatically process WBCs in a sample of blood for PCR amplification and CE analysis. A flow-through sample preparation procedure is used that does not require vortex-mixing, precipitation, and centrifugation, and is amenable to integration into a microfluidic circuit. TH01, a well-characterized microsatellite locus, served as the model for this study. TH01, a gene for human tyrosine hydroxylase, is a 4-bp repeat and comprises seven alleles (16).
Samples of whole blood collected from seven individuals were obtained from Stanford Blood Bank (Palo Alto, CA). The blood (
10 mL) was drawn into tubes containing citrate as the anticoagulant and then stored in a refrigerator until processed on a cartridge fluidic system. For each cartridge run, sample (200 µL), isotonic solution (6.5 mL), and lysis buffer (600 µL of 10 mmol/L Tris, 150 mmol/L NaCl, 1 mL/L Triton X-100, pH 8.5) were loaded in the appropriate chambers in the cartridge (Fig. 1A
). The cartridge contained a WBC extraction chamber that harbored a Leukosorb filter (Pall Corp) and a flexible membrane that interfaced with an ultrasonic transducer. The extraction chamber had a sweepable volume of 100 µL. The blood was pumped pneumatically through the filter at a rate of 0.5 mL/min. WBCs were collected on the filter, whereas all other material (i.e., red blood cells and plasma) passed through to a waste chamber. The trapped WBCs were washed with the isotonic solution, and then 200 µL of lysis buffer was pumped into the extraction chamber to displace the isotonic buffer. The WBCs were lysed on the filter by sonication for 15 s at 47 kHz. Lysis buffer (400 µL) was pumped into the extraction chamber, displacing the WBC lysate out to another chamber. The WBC lysate was manually collected and subjected to PCR and CE analysis. Six runs on the cartridge system were performed for each sample and were compared with runs using the QIAamp DNA Blood Mini Kit (Qiagen) as a reference. The cartridge run time was 9 min/sample.
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PCR amplification was accomplished on the Smart Cycler (Cepheid) using 25-µL reactions containing 1x TH01 STR (Fluorescein) primer pair (Promega), 1x STR buffer (Promega), 0.05 U/µL Platinum Taq DNA polymerase (Life Technologies), 1x Smart Cycler additive (0.2 g/L bovine serum albumin, 150 mmol/L trehalose, 2 mL/L Tween 20), and 2.5 µL of WBC lysate obtained from the cartridge. The thermal cycling conditions were as follows: an initial hold at 90 °C for 30 s; 10 cycles of 94 °C for 60 s, 60 °C for 30 s, and 70 °C for 45 s; 20 cycles of 90 °C for 30 s, 60 °C for 30 s, and 70 for 45 s; and a final hold at 60 °C for 60 s. Because the Smart Cycler exhibited fast ramp rates and precise temperature control, the cycling time was reduced to 55 min compared with 2.5 h on a conventional thermal cycler. CE was accomplished using the P/ACE MDQ system (Beckman) with a fused-silica capillary (ABI) filled with POP4 polymer (ABI) and 10x CE Buffer (ABI). PCR product was injected electrokinetically for 30 s at 5.0 kV. Separation and detection were performed for 15 min at 9.0 kV at 25 °C.
The mean amount of DNA collected from a 200-µL aliquot of blood processed on the cartridge system was 2.24 µg. A WBC extraction efficiency of 51% was calculated based on the relative yield of DNA obtained using the QIAamp Kit. The lower yield was partially attributable to a 25% loss of intact WBCs during storage of the blood (only intact WBCs are trapped by the filter). A 2.5-µL portion of the sample processed on the cartridge system or QIAamp Kit was subjected to PCR to amplify the TH01 locus. Agarose gel electrophoresis results (Fig. 1B
) indicated that the relative yield of PCR products obtained from samples prepared using the two methods were similar.
Allelic typing was performed by adding TH01 allelic ladder (Promega) to the PCR products generated from the WBC lysates and subjecting the mixtures to CE analysis (Fig. 1C
). The ladder ranged in size from 179 to 203 bp and consisted of the alleles designated 5, 6, 7, 8, 9, 9.3/10, and 11. The sharp, distinct TH01 allelic peaks observed on the electropherograms indicated that robust PCR amplification and CE separation analysis were achieved on the blood samples processed using the cartridge system. The PCR products exhibited a CE migration time of 13 min. The total time for processing, amplifying, and analyzing the blood sample was 84 min.
In summary, a cartridge system was used to process a 200-µL sample of whole blood in 9 min for genetic testing. This flow-through sample preparation procedure concentrated, extracted, and lysed WBCs from the blood without requiring vortex-mixing, precipitation, and centrifugation. The quantity and quality of DNA from the WBC lysate automatically prepared on the cartridge were sufficient for PCR and CE analysis of the TH01 STR locus, and the cartridge procedure was at least three times faster than the QIAamp Kit.
References
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