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Implementation of Laboratory Automation for the Analysis of STR Loci

STR loci are polymorphic genetic markers routinely used in forensic casework, parentage analysis and the construction of felon databases. The PowerPlexÔ system amplifies eight different loci simultaneously that can be detected by using two different fluorescent labels (Fluorescein tagged Gamma STRÔ loci D16S539, D7S820, D13S317, and D5S818 and the carboxy-tetramethylrhodamine tagged STR loci CSF1PO, TPOX, TH01, and vWA). Analysis of the eight loci contained in the Promega PowerPlexÔ system results in an average match probability exceeding one in one hundred million and an average power of exclusion greater than 99.7% (1). Even though STR methods are less labor intensive than RFLP methods, further operational efficiencies for STRs can be achieved through automation (2). In order to improve operational efficiencies while maintaining quality, we have automated the STR procedure and implemented the following systems:

· Accessioning of blood using the Rosys Gene Machine and bar code reader.

· The isolation of DNA utilizing a combination of the Fitzco FTAÔ paper and the Rosys robotic workstation.

· The use of multiplexed STR systems (Promega PowerPlexÔ: CSF1PO, TPOX, TH01 VWA, D16S539, D7S820, D13S317 , D5S818 and the Multiplex FFFL: F13A, F13B, VWA, and LPL,)

· Analysis of Results on the Hitachi FMBIO II^®

The purpose of this study was to validate and implement the use of the PowerPlexÔ, Fitzco FTAÔ Paper, and Rosys workstation in our laboratory. The robotic system was chosen based on its specimen throughput, precision, and ability to accommodate a plate washer, incubator and a microplate-hole punch device. The isolation of DNA from the Fitzco FTAÔ paper was validated based on the consistent quality and quantity of amplified product resulting from the purification of DNA. The ability to accurately amplify all of the observable alleles for a specific locus was part of the criterion for validating the PowerPlexÔ and the use of the FTAÔ protocol in our laboratory. Contamination studies were done to evaluate isolation and amplification of DNA. The final analysis of the STR products after electrophoresis was performed on the Hitachi FMBIO II^®. Validation of the Hitachi FMBIO II^® was previously presented (3,4).

Unless specifically stated DNA was isolated using the Promega Wizard^® Kit (5) or by using the following modification of the Fitzco FTAÔ protocol.

Five microliters of blood were spotted onto the FTAÔ paper and allowed to dry for one hour at room temperature. A one millimeter punch from the paper was placed into the well of a 96 well microtiter plate and 200 microliters of the Fitzco Purification reagent was added. The plate was incubated at room temperature for 5 minutes after which the buffer was removed. This procedure was repeated two times. After removing the third addition of Purification reagent, 200 microliters of 10 mM Tris, pH 8.0 was added. The plate was allowed to sit at room temperature for five minutes and then the reagent was removed. This procedure was repeated once. The final 10 mM Tris buffer was removed and the plate was dried at room temperature for at least an hour or for thirty minutes at 56^oC. The 1 mm punch containing the purified DNA was placed into a PCR reaction tube and amplified in a final reaction volume of 25 microliters. The purified DNA punch represented 2.5 microliters of the solution. The protocol for the amplification of the PowerPlexÔ system, CTTv and FFFL quadriplex system is described in the "Amplification Protocols".

Five microliters of blood were transferred to each well of the Fitzco FTAÔ Plate which is a 96 well microtiter plate containing the FTAÔ paper. The blood on the FTAÔ plate was dried in the Rosys incubator for forty-five minutes at 56^oC. Following drying, the plate was transferred to the vacuum assisted wash station for purification of the DNA. Utilizing the appropriate Rosys procedural script, the following purification steps were performed for each blood spot sample:

Following the purification steps and the ethanol wash, the plate was transferred to the incubator and dried for 45 minutes at 56^o C. After drying, a 1mm punch from the FTAÔ paper was delivered into a Robbins Scientific 96 well amplification plate using the Rosys gene machine integrated punching station. The 1mm punch containing the purified blood spot was amplified in a final reaction volume of 25 microliters (The 1mm blood punch replaces 2.5 micro liters of solution.) The Robbins 96 well amplification plate was sealed with plate caps and manually placed in the Perkin-Elmer 9600 thermal cycler. The protocol for the amplification of the PowerPlexÔ systems, FFFL, and CTTv quadriplex system is described in the "amplification protocol" section.

For DNA isolated from liquid blood, an average of twenty nanograms of genomic DNA was amplified using the Perkin-Elmer 9600 following the protocol described in the Promega Technical Manuals for the PowerPlexÔ System or the GenePrintÔ Fluorescent quadriplex systems (6,7). For DNA isolated from dried blood stains on Fitzco FTAÔ paper, a 1 millimeter punch from a five microliter spot of blood was amplified. DNA was not quantified from dried blood stains on FTAÔ paper. Amplified Multiplex product was electrophoretically separated in a 32 cm denatured 6% polyacrylamide gel on a Life Technologies SA32 gel box. The gel was pre-run (using 1X TBE) for 15 minutes at 30 watts constant power. The samples were mixed with tracking dye, heat denatured for 2 minutes at 95^oC, loaded onto the gel, and electrophoresed at 30 watts for approximately 210 minutes at constant power. Appropriate negative and positive controls were included for every amplification reaction and loaded onto the gels along with the appropriate allelic ladders from the Promega STR kits.

Following electrophoresis of the samples, the gels were analyzed on the Hitachi FMBIO II^®. For the PowerPlexÔ systems the Gamma STRÔ loci (D16S539, D7S820, D13S317, and D5S818) were scanned at 505 nm and the carboxy-tetramethylrhodamine (TMR) labeled primers CSF1PO, TPOX, TH01, and vWA (CTTv) were analyzed at the 585 scan. The GenePrintÔ quadriplex fluorescein labeled primers CTTv and F13A01, FESFPS, F13B, and LPL (FFFL) were scanned at 505 as described in the manual (6). Specimen genotypes were determined using the Hitachi FMBIO^® software which converts the molecular base-pair sizes to alleles descriptions based on the tandem repeat number.

The Promega PowerPlexÔ system was evaluated with regards to its accuracy, consistency, sensitivity and ability to amplify DNA from different extraction methods as well as from different biological substrates.

Storage, purification and amplification of DNA purified from blood spotted onto FTAÔ paper was validated with regards to the accuracy and consistency of results, ability of the DNA to be amplified from a variety of primers, sensitivity of the reaction, and potential contamination of the DNA.

The procedure for storing blood and purifying DNA using the FTAÔ paper was validated for the following parameters.

The Rosys Plato Gene Machine was utilized to integrate the use of the FTAÔ paper in the amplification of the DNA. The Rosys instrument was used to semi-automate the dispensing of blood onto FTAÔ paper, purify the DNA from the blood, punch the FTAÔ paper into the 96 well amplification plate, and distribute the amplification reaction mixture.

The processing of samples for analysis by STR technology can become more efficient by the purification of DNA from Fitzco FTAÔ paper and amplification of the DNA using the Promega PowerPlexÔ system. The Fitzco FTAÔ paper permits one to store blood at room temperature at length and allows one a convenient and efficient method to purify DNA. The purified DNA is bound to the FTAÔ paper and presents itself as a template for amplification. Storage and processing of the blood to purified DNA using the FTAÔ purification process has been shown to be an accurate, reliable process which is free of potential contamination. In our laboratory, the manual processing of DNA for amplification using FTAÔ paper has reduced the amount of labor time by 40% as compared to extraction by phenol chloroform or salt precipitation.

The PowerPlexÔ system amplifies eight different loci simultaneously and can be detected by two different fluorescent dyes (Fluorescein tagged Gamma STRÔ loci D16S539, D7S820, D13S317, and D5S818 and the carboxy-tetramethylrhodamine STR loci CSF1PO, TPOX, TH01, and vWA). These validation studies have shown that the PowerPlexÔ is accurate, consistent, sensitive below 1 nanogram of DNA and versatile with regards to the method of isolation and original biological substrate. Amplification of 8 loci and analysis of the products in one gel lane with the use of the Hitachi FMBIO II^® decreases labor time considerably by minimizing the number of gels that a technician must prepare. Also, fewer pipetting steps are required during amplification and gel loading.

Incorporation of these procedures with a semi-automated processing of the samples with the Rosys Gene Machine has been shown to produce accurate and reliable results with improved operational efficiencies. At this time the only technical setback is the punching of the FTAÔ paper into the wells. Once this problem is solved it is anticipated that the dispensing of blood, storage of blood, purification of DNA, and addition of amplification reagents will become an automated process which will allow the lab to handle at least 200 samples per day. Automation will assist in the processing of large number of samples on a daily basis for the building of DNA databases or for the processing of paternity casework. This validation study has demonstrated that automation will greatly enhance the laboratory efficiency and still maintain the quality of results as accurate and reliable.

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